Biomarkers predictive of therapeutic responsiveness to chimeric antigen receptor therapy and uses thereof

ABSTRACT

Cancer biomarkers and methods of using them are disclosed.

This application is a continuation of U.S. application Ser. No.15/517,597, filed Apr. 7, 2017, now allowed, which is a U.S. nationalphase application under 35 U.S.C. § 371 of International Application No.PCT/US2015/054542, filed Oct. 7, 2015, which claims priority to U.S.Ser. No. 62/061,553 filed Oct. 8, 2014 and U.S. Ser. No. 62/144,682filed Apr. 8, 2015, the contents of which are incorporated herein byreference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 7, 2015, isnamed N2067-7057WO_SL.txt and is 219,221 bytes in size.

FIELD OF THE INVENTION

The invention relates to cancer biomarkers and uses thereof.

BACKGROUND OF THE INVENTION

Many patients with B cell malignancies are incurable with standardtherapy. In addition, traditional treatment options often have seriousside effects. Attempts have been made in cancer immunotherapy, however,several obstacles render the goal of clinical effectiveness difficult toachieve. Although hundreds of so-called tumor antigens have beenidentified, these are generally derived from self and thus are poorlyimmunogenic. Furthermore, tumors use several mechanisms to renderthemselves hostile to the initiation and propagation of immune attack.

Recent developments using chimeric antigen receptor (CAR) modifiedautologous T cell (CART) therapy, which relies on redirecting T cells toa suitable cell-surface molecule on cancer cells such as B cellmalignancies, show promising results in harnessing the power of theimmune system to treat B cell malignancies and other cancers (see, e.g.,Sadelain et al., CANCER DISCOVERY 3:388-398 (2013)). For example, theclinical results of a CART that binds to CD19 (i.e., “CTL019”) haveshown promise in establishing complete remissions in patients sufferingwith chronic lymphocytic leukemia (CLL), as well as in childhood acutelymphocytic leukemia (ALL) (see, e.g., Kalos et al., SCI TRANSL MED3:95ra73 (2011), Porter et al., NEJM 365:725-733 (2011), Grupp et al.,NEJM 368:1509-1518 (2013)).

Besides the ability for the chimeric antigen receptor on the geneticallymodified T cells to recognize and destroy the targeted cells, asuccessful therapeutic T cell therapy needs to have the ability toproliferate, to persist over time, and to further monitor for leukemiccell escapees. The variable phenotypic state of T cells, whether it isin a state of anergy, suppression or exhaustion, will have effects onCAR-transformed T cells' efficacy. To be effective, CAR transformedpatient T cells need to persist and maintain the ability to proliferatein response to the CAR's antigen.

A need, therefore, exists for a method of using biomarkers for use inconnection with the differential diagnosis and treatment of cancer withCAR-expressing cell (e.g., T cell, NK cell) therapy. In particular,there is an unmet need for effective predictors of therapeutic responsein subjects having a hematological cancer, such as CLL and ALL, to aCAR-expressing cell therapy, e.g., with CTL019 or other CD19CAR-expressing cells.

SUMMARY OF THE INVENTION

The present disclosure relates to the identification and use ofanalytes, analyte profiles, or markers (e.g., gene expression, flowcytometry and/or protein expression profiles) with clinical relevance tocancer (e.g., a hematological cancer such as chronic lymphocyticleukemia (CLL) and acute lymphocytic leukemia (ALL)). In someembodiments, the disclosure provides the identity of genes, whoseexpression, at the transcriptional and protein levels, are correlatedwith CLL and ALL progression, e.g., as a way of predicting a response toa Chimeric Antigen Receptor (CAR)-expressing cell therapy (e.g., atherapy comprising a cell (e.g., an immune effector cell or populationof cells) that expresses a CAR that binds to CD19 (also referred toherein as a “CAR19” or “CD19 CAR”-expressing cell). In certainembodiments, one or more of a CD19 CAR-expressing cell gene setsignature, a biomarker listed in Table 1A, Table 1B, Table 7A, Table 7B,Table 8, Table 9, Table 10, Table 14, Table 15, Table 16 (e.g., CCL20,IL-17a and/or IL-6), Table 17, Table 18, Table 20, a CD27 biomarker, aCD45RO biomarker, a PD-1 biomarker, a LAG-3 biomarker, a TIM-3biomarker, an IL2RA biomarker, an IL21 biomarker, a CD4 biomarker, a CD8biomarker, a TH1+ helper T cell gene set signature, a TH2+ helper T cellgene set signature, a memory T cell (e.g., a CD8+ memory T cell, e.g., anaïve T cell (T_(N)), e.g. a memory stem cell (T_(SCM)), e.g. a centralmemory T cell (T_(CM)), e.g. an effector memory T cell (T_(EM))) geneset signature, and combinations thereof) are evaluated. These geneexpression profiles may be applied to the diagnosis and/or prognosis ofa cancer, e.g., a hematological cancer such as CLL and ALL, and areparticularly useful in predicting whether a subject will respondfavorably to a CAR therapy (e.g., a CD19 CAR therapy as described here,e.g., a CTL019 therapy) in a subject diagnosed with a cancer, e.g., ahematological cancer such as CLL or ALL. Compared to clinical parametersor biochemical markers used in existing prognosis methods, theexpression profiles of the genes disclosed herein constitute a morerobust signature of hematological cancer progression (e.g., CLL and ALLprogression) and provide a more reliable, non-subjective basis for theselection of appropriate therapeutic regimens.

Amongst other things, the present disclosure provides novel genesignatures, e.g., at the transcriptional and protein levels, and methodsof use thereof, that predict subject response to a cell expressing aCAR, e.g., a CD19 CAR (e.g., a CD19 CAR-expressing cell, e.g., T cell,NK cell, described herein such as, e.g., CTL019) therapy in a cancer,e.g., a hematological cancer such as CLL and ALL.

The present disclosure demonstrates, at least in part, that expressionprofiles and gene signatures, e.g., at the transcriptional and proteinlevels, are useful to distinguish among a responder, a partialresponder, a non-responder, a relapser or a non-relapser to a therapycomprising a CAR-expressing cell (e.g., a CAR-expressing immune effectorcell, e.g., a T cell, or an NK cell), (also referred to herein as a“CAR-expressing cell therapy”), in a cancer (e.g., a hematologicalcancer such as CLL and ALL). In one embodiment, the CAR-expressing cellis a CD19 CAR-expressing cell. In one embodiment, the therapy is aCTL019 therapy. In embodiments, the expression profiles and genesignatures disclosed herein distinguish among a CAR (or CD19CAR)-expressing cell responder, a CAR (or CD19 CAR)-expressing cellpartial responder, or a CAR (or CD19 CAR)-expressing cell non-responder(e.g., a CTL019-responder, a CTL019-partial responder, and aCTL019-non-responder); or a CAR (or CD19 CAR)-expressing cell relapser,or a CAR (or CD19 CAR)-expressing cell non-relapser (e.g., aCTL019-relapser, or a CTL019-relapser), in a cancer (e.g., ahematological cancer such as CLL and ALL). The present disclosureencompasses the identification of novel gene signatures predictive ofsubject response to a CAR-expressing cell therapy, e.g., a CD19CAR-expressing cell therapy such as CTL019.

Thus, disclosed herein are methods, systems, compositions, and kits forthe identification, assessment and/or treatment of a subject havingcancer. Exemplary cancers include, but are not limited to, B-cell acutelymphocytic leukemia (B-ALL), T-cell acute lymphocytic leukemia (T-ALL),acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML),chronic lymphocytic leukemia (CLL), B cell promyelocytic leukemia,blastic plasmacytoid dendritic cell neoplasm, Burkitt

lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cellleukemia, small cell- or a large cell-follicular lymphoma, malignantlymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma(MCL), marginal zone lymphoma, multiple myeloma, myelodysplasia andmyelodysplastic syndrome, non-Hodgkin lymphoma, Hodgkin lymphoma (HL),plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, andWaldenstrom macroglobulinemia. In one embodiment, the cancer is ALL. Inanother embodiment, the cancer is CLL. In an embodiment, the cancer isassociated with CD19 expression.

Accordingly, in one aspect, the invention features a method ofevaluating a subject having a cancer, e.g., a hematological cancer. Themethod includes acquiring a value of responder or relapser status (e.g.,a value of responder or relapser status as described herein) to atherapy comprising a CAR-expressing cell (e.g., a plurality (e.g., apopulation) of CAR (e.g., CAR19-)-expressing cells) for the subject,wherein said value is indicative of the subject's responsiveness orrelapsing status to the CAR-expressing cell therapy.

In a related aspect, the invention features a method of evaluating ormonitoring the effectiveness of a CAR-expressing cell therapy in asubject having a cancer, e.g., a hematological cancer. The methodincludes acquiring a value of responder or relapser status (e.g., avalue of responder or relapser status as described herein) to a therapycomprising a CAR-expressing cell (e.g., a plurality (e.g., a population)of CAR (e.g., CAR19-)-expressing cells) for the subject, wherein saidvalue is indicative of the effectiveness of the CAR-expressing celltherapy, thereby evaluating the effectiveness of the CAR-expressing celltherapy in the subject.

In another aspect, the invention features a method for treating asubject having a cancer, e.g., a hematological cancer. The methodincludes administering to the subject a therapeutically effective doseof a CAR-expressing cell therapy, if the subject is identified as beingresponsive (e.g., identified as a complete responder, partial responderor a non-relapser) to a therapy comprising a CAR-expressing cell (e.g.,a plurality (e.g., a population) of CAR (e.g., CAR19-)-expressingcells), wherein said identifying comprises a value of responder orrelapser status (e.g., a value of responder or relapser status asdescribed herein).

In a related aspect, the invention features a method of treating acancer, e.g., a hematological cancer, in a subject. The method includesacquiring a value of responder or relapser status (e.g., a value ofresponder or relapser status as described herein) to a therapycomprising a CAR-expressing cell (e.g., a plurality (e.g., a population)of CAR (e.g., CAR19-)-expressing cells) for the subject; and responsiveto said value, treating the cancer.

In embodiments of any of the methods and compositions for use describedherein, the value of responder or relapser status comprises a measure ofone, two, three, four, five, six, seven or more (all) of the following:

(i) the level or activity of CD27 and/or CD45RO− (e.g., CD27+CD45RO−)immune effector cells, e.g., in a CD4+ or a CD8+ T cell population, in asubject, e.g., a sample from the subject (e.g., an apheresis sample or aCAR-expressing cell product sample);

(ii) the level or activity of one, two, three, or more (e.g., all) ofresting T_(EFF) cells, resting T_(REG) cells, younger T cells (e.g.,younger CD4 or CD8 cells, or gamma/delta T cells), or early memory Tcells, or a combination thereof, in a subject, e.g., a sample from thesubject (e.g., an apheresis sample or a CAR-expressing cell productsample);

(iii) the level or activity of one, two, three, or more (e.g., all) ofactivated T_(EFF) cells, activated T_(REG) cells, older T cells (e.g.,older CD4 or CD8 cells), or late memory T cells, or a combinationthereof, in a subject, e.g., a sample from the subject (e.g., anapheresis sample or a manufactured CAR-expressing cell product sample);

(iv) the level or activity of an immune cell exhaustion marker, e.g.,one, two or more immune checkpoint inhibitors (e.g., PD-1, TIM-3 and/orLAG-3) in a subject, e.g., a sample from the subject (e.g., an apheresissample or a manufactured CAR-expressing cell product sample);

(v) the level or activity of one, two, three, four, five, ten, twenty ormore of the biomarkers listed in Table 1A, Table 1B, Table 7A, Table 7B,Table 8, Table 9, Table 10, Table 14 (e.g., CCL20, IL-17a and/or IL-6),Table 16, Table 17, Table 18, Table 20, FIG. 2B, PD-1, LAG-3, TIM-3,CD57, CD27, CD122, CD62L, KLRG1, or a CD19 CAR-expressing cell gene setsignature;

(vi) a cytokine level or activity (e.g., quality of cytokine reportoire)in a CAR-expressing cell product sample, e.g., CAR19-expressing cellproduct sample (e.g., CTL019), wherein the cytokine is chosen from one,two, three, four, five or more (or all) of the cytokines listed in Table16;

(vii) a transduction efficiency of a CAR-expressing cell in aCAR-expressing cell product sample; or

(viii) a quantity of CD27+PD-1− cells in a subject, e.g., a sample fromthe subject (e.g., an apheresis sample or a CAR-expressing cell productsample, e.g., CAR19-expressing cell product sample (e.g., CTL019)),e.g., a quantity greater than or equal to 1×10⁷ cells.

In an aspect, the invention provides a CAR expressing cell therapy(e.g., CD19 CART cell, e.g., CTL019 cell) for use in the treatment of asubject, wherein the CAR expressing cell has been assayed according to amethod herein, e.g., before or after transduction or transfection with aCAR nucleic acid. In a related aspect, the invention provides a CARexpressing cell therapy (e.g., CD19 CART cell, e.g., CTL019 cell), foruse in the treatment of a subject that has been identified as beingresponsive (e.g., identified as a complete responder, partial responderor a non-relapser) to a therapy comprising a CAR-expressing cellpopulation (e.g., a CAR19-expressing cell population). The compositionfor use can comprise a measure of one, two, three, four, five, six,seven, or more (all) of (i)-(viii) described herein.

Alternatively, or in combination with the methods and compositions foruse disclosed herein, responsive to said value, performing one, two,three, four, five, six, seven, or more (e.g., all) of:

identifying the subject as a complete responder, partial responder ornon-responder, or a relapser or a non-relapser;

administering e.g., to a responder or a non-relapser, a CAR-expressingcell therapy;

administering an altered dosing of a CAR-expressing cell therapy;

altering the schedule or time course of a CAR-expressing cell therapy;

administering, e.g., to a non-responder or a partial responder, anadditional agent in combination with a CAR-expressing cell therapy,e.g., a checkpoint inhibitor, e.g., a checkpoint inhibitor describedherein;

administering to a non-responder or partial responder a therapy thatincreases the number of younger T cells in the subject prior totreatment with a CAR-expressing cell therapy;

modifying a process, e.g., a manufacturing process, of a CAR-expressingcell therapy, e.g., enriching for younger T cells prior to introducing anucleic acid encoding a CAR, or increasing the transduction efficiency,e.g., for a subject identified as a non-responder or a partialresponder;

administering an alternative therapy, e.g., for a non-responder orpartial responder or relapser, e.g., a standard of care for a particularcancer type; or

if the subject is, or is identified as, a non-responder or a relapser,decreasing the T_(REG) cell population and/or T_(REG) gene signature,e.g., by one or more of CD25 depletion, or administration ofcyclophosphamide, anti-GITR antibody, an mTOR inhibitor, or acombination thereof.

In certain embodiments, the subject is pre-treated with an anti-GITRantibody. In certain embodiment, the subject is treated with ananti-GITR antibody prior to infusion or re-infusion. In someembodiments, the subject is a patient with CLL.

In another aspect, the invention features a method of, or assay for,identifying a subject having a cancer as having an increased ordecreased likelihood to respond to a treatment that comprises aCAR-expressing cell (e.g., a plurality (e.g., a population) of CAR(e.g., CAR19-)-expressing cells). The method includes:

(1) providing, e.g., acquiring, a sample from the subject;

(2) determining a level or activity of one or more biomarkers listed inTable 1A, Table 1B, Table 7A, Table 7B, Table 8, Table 9, Table 10,Table 14 (e.g., CCL20, IL-17a and/or IL-6), Table 17, Table 18, Table20, PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L, KLRG1 in the sample;

wherein a difference, e.g., a statistically significant difference,between the determined level compared to a reference level is predictiveof the subject's responsiveness to the CAR-expressing cell therapy; and

(3) (optionally) identifying the subject as a complete responder,partial responder, non-responder, a relapser or a non-relapser, to theCAR-expressing cell therapy.

In yet another aspect, the invention features a method for treating asubject having a cancer comprising:

determining if the subject has an increased likelihood to respond, or adecreased likelihood to relapse, to a CAR-expressing cell therapy (e.g.,a CAR19-expressing therapy, e.g., CTL019) by determining the level oractivity of one or more biomarkers in Table 1A, Table 1B, Table 7A,Table 7B, Table 8, Table 9, Table 10, Table 14 (e.g., CCL20, IL-17aand/or IL-6), FIG. 2B, Table 17, Table 18, Table 20, PD-1, LAG-3, TIM-3,CD57, CD27, CD122, CD62L, KLRG1, or a CD19 CAR-expressing cell gene setsignature in a sample from the subject, e.g., relative to a referencelevel; and administering to the subject a therapeutically effective doseof a CAR-expressing cell therapy.

In yet another aspect, the invention features a method for treating asubject having cancer comprising:

(1) determining if the subject has an increased likelihood to relapse toa CAR-expressing cell therapy by acquiring a value for the level oractivity of one or more markers in a Table herein, e.g., Table 17 in asample from the subject (e.g., an apheresis sample or a manufacturedCAR-expressing product sample), wherein a difference, e.g., astatistically significant difference, in the level or activity of one ormore biomarker genes relative to the reference level is indicative of anincreased likelihood of relapse to a CAR-expressing cell therapy; and

(2) for a subject with an increased likelihood of relapse, decreasingthe T_(REG) cell population and/or decreasing T_(REG) gene signature;and

(3) administering to the subject a therapeutically effective dose of aCAR-expressing cell therapy.

Additional features and embodiments of the present invention include oneor more of the following:

In some embodiments of any of the methods and compositions for usedisclosed herein, an immune cell has an exhausted phenotype, e.g.,co-expresses at least two exhaustion markers, e.g., co-expresses PD-1and TIM-3. In other embodiments, an immune cell has an exhaustedphenotype, e.g., co-expresses at least two exhaustion markers, e.g.,co-expresses PD-1 and LAG-3.

In some embodiments of any of the methods, systems, compositions foruse, and kits disclosed herein, the CAR-expressing cell therapycomprises a plurality (e.g., a population) of CAR-expressing immuneeffector cells, e.g., a plurality (e.g., a population) of T cells or NKcells, or a combination thereof. In one embodiment, the CAR-expressingcell therapy is a CAR19 therapy (e.g., CTL019 therapy). In anembodiment, the CAR-expressing cell therapy comprises or consists ofCTL019. In an embodiment, the CAR-expressing cell is a CTL019 product.In an embodiment, the CAR-expressing cell is a T cell. In an embodiment,the CAR-expressing cell is a NK cell.

In some embodiments of any of the methods and compositions for usedisclosed herein, the measure of one or more of (i)-(viii) is obtainedfrom an apheresis sample acquired from the subject. The apheresis samplecan be evaluated prior to infusion or re-infusion.

In some embodiments of any of the methods and compositions for usedisclosed herein, the measure of one or more of (i)-(viii) is obtainedfrom a manufactured CAR-expressing cell product sample, e.g.,CAR19-expressing cell product sample (e.g., CTL019). The manufacturedCAR-expressing cell product can be evaluated prior to infusion orre-infusion.

In some embodiments of any of the methods and compositions for usedisclosed herein, the subject is evaluated prior to receiving, during,or after receiving, the CAR-expressing cell therapy.

In some embodiments of any of the methods and compositions for usedisclosed herein, the hematological cancer is an ALL or a CLL. Thesubject can be a human patient.

In some embodiments of any of the methods and compositions for usedisclosed herein, the cell, e.g., the population of immune effectorcells (e.g., cells expressing a CAR molecule described herein) isadministered in combination with an inhibitor of an immune checkpointmolecule chosen from one or more of PD1, PD-L1, PD-L2, CTLA4, TIM3,CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA,TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM(TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9,adenosine, TGFR (e.g., TGFR beta), or a combination thereof.

In some embodiments of any of the methods and compositions for usedisclosed herein, the subject receives concurrent treatment with anagent, e.g., an mTOR inhibitor, and/or a checkpoint inhibitor. In someembodiments, the subject receives treatment with an agent, e.g., an mTORinhibitor, and/or a checkpoint inhibitor, post-CAR-expressing celltherapy. In some embodiments, the subject receives a pre-treatment ofwith an agent, e.g., an mTOR inhibitor, and/or a checkpoint inhibitor,prior to the initiation of a CAR-expressing cell therapy.

In some embodiments of any of the methods and compositions for usedisclosed herein, T_(REG) cell population and/or T_(REG) gene signatureis decreased prior to collection of cells for manufacturing. In someembodiments, the T_(REG) cell population and/or T_(REG) gene signatureis decreased prior to CAR-expressing cell (e.g., T cell, NK cell)therapy. In some embodiments, the T_(REG) cell population and/or T_(REG)gene signature is decreased by administration of cyclophosphamide,anti-GITR antibody, an mTOR inhibitor, or a combination thereof.

In some embodiments of any of the methods and compositions for usedisclosed herein the value of responder or relapser status comprises ameasure of a combination of a gene signature and a biomarker. In someembodiments, the value of the responder or relapser status comprises ameasure of a CD19 CAR-expressing cell gene set signature and acombination of one or more of: a biomarker listed in Table 1A, Table 1B,Table 7A, Table 7B, Table 8, Table 9, Table 10, Table 14 (e.g., CCL20,IL-17a and/or IL-6), Table 17, Table 18, Table 20, PD-1, LAG-3, TIM-3,CD57, CD27, CD122, CD62L, or KLRG1.

In some embodiments of any of the methods and compositions for usedisclosed herein, the method further comprises identifying the subjectas a responder (e.g., a complete or partial responder), a non-responder,a relapser or a non-relapser, based on a measure of one or more of(i)-(viii).

In some embodiments of any of the methods and compositions for usedisclosed herein, the measure of one or more of (i)-(viii) evaluates aprofile for one or more of gene expression, flow cytometry or proteinexpression.

In some embodiments of any of the methods and compositions for usedisclosed herein, the expression profile includes one or more genesignatures based on mRNA expression levels of selected genes obtainedfrom the apheresis sample or a manufactured CD19 CAR-expressing cellproduct (e.g., CTL019). In one embodiment, the expression profileincludes one, two, three, four, five, ten, twenty or more of a biomarkerlisted in Table 1A, Table 1B, Table 7A, Table 7B, Table 8, Table 9,Table 10, Table 14 (e.g., CCL20, IL-17a and/or IL-6), Table 16, Table17, Table 18, Table 20, FIG. 2B, PD-1, LAG-3, TIM-3, CD57, CD27, CD122,CD62L, KLRG1, or a CD19 CAR-expressing cell gene set signature.

In some embodiments of any of the methods and compositions for usedisclosed herein, the level or activity of a CD8+ T cell is evaluatedusing a profile or signature indicative of the percentage of CD8+ T cellin the sample.

In some embodiments of any of the methods and compositions for usedisclosed herein, the level or activity of CD27+CD45RO− immune effectorcells is evaluated using a profile or signature indicative of thepercentage of CD27+CD45RO− immune effector cells in the sample.

In some embodiments of any of the methods and compositions for usedisclosed herein, the level or activity, e.g., in (i), (ii), or (v), isevaluated using a profile or gene signature according to one, two,three, four, five, ten, twenty, fifty, sixty, seventy, one hundred ormore of a biomarker or gene set listed in Tables 1A, 1B, 3, 4, 5, 6, orFIG. 2B.

In some embodiments of any of the methods and compositions for usedisclosed herein, the level or activity one, two or more immunecheckpoint inhibitors is evaluated, e.g., using flow cytometry, as anindicator of the percentage of PD-1+/LAG-3+ cells in the CAR-expressingcell population (e.g., a CAR19+ cell population).

In some embodiments of any of the methods and compositions for usedisclosed herein, the level or activity one, two or more immunecheckpoint inhibitors is evaluated, e.g., using flow cytometry, as anindicator of the percentage of PD-1+/TIM-3+ cells in the CAR-expressingcell population (e.g., a CAR19+ cell population).

In some embodiments of any of the methods and compositions for usedisclosed herein, the level or activity of one, two, three, four, five,ten, twenty, fifty, sixty, seventy, one hundred or more of a biomarkeror gene set listed in Table 7A, Table 7B, Table 8 and FIG. 2B predicts asubject's response to a CAR19+ cell product (e.g., CTL019).

In some embodiments of any of the methods and compositions for usedisclosed herein, the value of responder or relapser status comprises ameasure of the level or activity of one, two, three, four, five, ten,twenty or more (e.g., all) of the biomarkers having a given FDR p-value,listed herein, e.g., in a Table herein. In some embodiments, the FDRp-value is below 0.2, 0.1, 0.05, 0.02, 0.01, 0.005, 0.002, or 0.001. Insome embodiments, the FDR p-value is below 0.1 or 0.01. In someembodiments, the biomarkers are biomarkers listed in Table 1A, Table 1B,Table 16, Table 17, Table 18, or Table 20, or a combination thereof. Insome embodiments, the measure comprises a measure of all of thebiomarkers in Table 1A that have a p-value below a threshold of 0.2,0.1, 0.05, 0.02, 0.01, 0.005, 0.002, or 0.001. In some embodiments, themeasure comprises a measure of all of the biomarkers in Table 1B thathave a p-value below a threshold of 0.2, 0.1, 0.05, 0.02, 0.01, 0.005,0.002, or 0.001. In some embodiments, the measure comprises a measure ofall of the biomarkers in Table 16 that have a p-value below a thresholdof 0.2, 0.1, 0.05, 0.02, 0.01, 0.005, 0.002, or 0.001. In someembodiments, the measure comprises a measure of all of the biomarkers inTable 17 that have a p-value below a threshold of 0.2, 0.1, 0.05, 0.02,0.01, 0.005, 0.002, or 0.001. In some embodiments, the measure comprisesa measure of all of the biomarkers in Table 18 that have a p-value belowa threshold of 0.2, 0.1, 0.05, 0.02, 0.01, 0.005, 0.002, or 0.001. Insome embodiments, the measure comprises a measure of all of thebiomarkers in Table 20 that have a p-value below a threshold of 0.2,0.1, 0.05, 0.02, 0.01, 0.005, 0.002, or 0.001. In some embodiments, themeasure comprises a measure of all of the biomarkers having a p-valuebelow the threshold. In some embodiments, the measure comprises ameasure of one, two, three, four, five, ten, twenty, fifty, or onehundred biomarkers having a p-value below the threshold. In someembodiments, the measure comprises a measure of at least one, two,three, four, five, ten, twenty, fifty, or one hundred biomarkers havinga p-value below the threshold. In some embodiments, the measurecomprises a measure of 1-5, 5-10, 10-20, 20-50, or 50-100 biomarkershaving a p-value below the threshold.

In some embodiments, biomarkers of Table 7B that are designated “CR” inthe table are upregulated in complete responders compared tonon-responders. In some embodiments, biomarkers of Table 7B that aredesignated “NR” in the table are upregulated in non-responders comparedto complete responders.

In some embodiments of any of the methods and compositions for usedisclosed herein, the biomarker is a secreted or a cell surfacebiomarker listed in Table 8. For example the biomarker can be measuredby flow cytometry.

In some embodiments of any of the methods and compositions for usedisclosed herein, a responder (e.g., a complete responder) has, or isidentified as having, a greater level or activity of one, two, or more(all) of GZMK, PPF1BP2, or naïve T cells as compared to a non-responder.In some embodiments of any of the methods and compositions for usedisclosed herein, a non-responder has, or is identified as having, agreater level or activity of one, two, three, four, five, six, seven, ormore (e.g., all) of IL22, IL-2RA, IL-21, IRF8, IL8, CCL17, CCL22,effector T cells, or regulatory T cells, as compared to a responder.

In an embodiment of any of the methods and compositions for usedisclosed herein, a relapser is a patient having, or who is identifiedas having, an increased level of expression of one or more of (e.g., 2,3, 4, or all of) the following genes, compared to non relapsers:MIR199A1, MIR1203, uc021ovp, ITM2C, and HLA-DQB1 and/or a decreasedlevels of expression of one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9,10, 11, or all of) the following genes, compared to non relapsers:PPIAL4D, TTTY10, TXLNG2P, MIR4650-1, KDMSD, USP9Y, PRKY, RPS4Y2, RPS4Y1,NCRNA00185, SULT1E1, and EIF1AY.

In some embodiments of any of the methods and compositions for usedisclosed herein, a complete responder has, or is identified as having,a greater, e.g., a statistically significant greater, percentage of CD8+T cells compared to a reference value, e.g., a non-responder percentageof CD8+ T cells.

In some embodiments of any of the methods and compositions for usedisclosed herein, a complete responder has, or is identified as having,a greater percentage (e.g., 5%, 6%, 7%, 10%, 15%, 20%, 25%, 27%, 30%,35%, or 40% or greater number) of CD27+CD45RO− immune effector cells,e.g., in the CD8+ population, compared to a reference value, e.g., anon-responder number of CD27+CD45RO− immune effector cells.

In some embodiments of any of the methods and compositions for usedisclosed herein, a complete responder or a partial responder has, or isidentified as having, a greater, e.g., a statistically significantgreater, percentage of CD4+ T cells compared to a reference value, e.g.,a non-responder percentage of CD4+ T cells.

In some embodiments of any of the methods and compositions for usedisclosed herein, a complete responder has, or is identified as having,a greater percentage of one, two, three, or more (e.g., all) of restingT_(EFF) cells, resting T_(REG) cells, younger T cells (e.g., younger CD4or CD8 cells, or gamma/delta T cells), or early memory T cells, or acombination thereof, compared to a reference value, e.g., anon-responder number of resting T_(EFF) cells, resting T_(REG) cells,younger T cells (e.g., younger CD4 or CD8 cells), or early memory Tcells.

In some embodiments of any of the methods and compositions for usedisclosed herein, a non-responder has, or is identified as having, agreater percentage of one, two, three, or more (e.g., all) of activatedT_(EFF) cells, activated T_(REG) cells, older T cells (e.g., older CD4or CD8 cells), or late memory T cells, or a combination thereof,compared to a reference value, e.g., a responder number of activatedT_(EFF) cells, activated T_(REG) cells, older T cells (e.g., older CD4or CD8 cells), or late memory T cells.

In some embodiments of any of the methods and compositions for usedisclosed herein, a non-responder has, or is identified as having, agreater percentage of an immune cell exhaustion marker, e.g., one, twoor more immune checkpoint inhibitors (e.g., PD-1, TIM-3 and/or LAG-3).In one embodiment, a non-responder has, or is identified as having, agreater percentage of PD-1 or LAG-3 expressing immune effector cells(e.g., CD4+ T cells and/or CD8+ T cells) (e.g., CAR-expressing CD4+cells and/or CD8+ T cells) compared to the percentage of PD-1 or LAG-3expressing immune effector cells from a responder.

In some embodiments of any of the methods and compositions for usedisclosed herein, a non-responder has, or is identified as having, agreater percentage of immune cells having an exhausted phenotype, e.g.,immune cells that co-express at least two exhaustion markers, e.g.,co-expresses PD-1 and TIM-3. In other embodiments, a non-responder has,or is identified as having, a greater percentage of immune cells havingan exhausted phenotype, e.g., immune cells that co-express at least twoexhaustion markers, e.g., co-expresses PD-1 and LAG-3.

In some embodiments of any of the methods and compositions for usedisclosed herein, a non-responder has, or is identified as having, agreater percentage of PD-1+/LAG-3+ cells in the CAR-expressing cellpopulation (e.g., a CAR19+ cell population) compared to a responder(e.g., a complete responder) to the CAR-expressing cell therapy.

In some embodiments of any of the methods and compositions for usedisclosed herein, a partial responder has, or is identified as having, ahigher percentages of PD-1+/LAG-3+ cells, than a responder, in theCAR-expressing cell population (e.g., a CAR19+ cell population).

In some embodiments of any of the methods and compositions for usedisclosed herein, a non-responder has, or is identified as having, anexhausted phenotype of PD1+CAR+ and co-expression of LAG3 in theCAR-expressing cell population (e.g., a CAR19+ cell population).

In some embodiments of any of the methods and compositions for usedisclosed herein, a non-responder has, or is identified as having, agreater percentage of PD-1+/TIM-3+ cells in the CAR-expressing cellpopulation (e.g., a CAR19+ cell population) compared to the responder(e.g., a complete responder).

In some embodiments of any of the methods and compositions for usedisclosed herein, a partial responders has, or is identified as having,a higher percentage of PD-1+/TIM-3+ cells, than responders, in theCAR-expressing cell population (e.g., a CAR19+ cell population).

In some embodiments of any of the methods and compositions for usedisclosed herein, the presence of CD8+CD27+CD45RO− T cells in anapheresis sample is a positive predictor of the subject response to aCAR-expressing cell therapy (e.g., a CAR19 therapy (e.g., CTL019therapy)).

In some embodiments of any of the methods and compositions for usedisclosed herein, a high percentage of PD1+CAR+ and LAG3+ or TIM3+ Tcells in an apheresis sample is a poor prognostic predictor of thesubject response to a CAR-expressing cell therapy (e.g., a CAR19 therapy(e.g., CTL019 therapy)).

In some embodiments of any of the methods and compositions for usedisclosed herein, a responder (e.g., a complete responder) to a CAR19therapy has, or is identified as having, the biomarker profile of Table9.

In some embodiments of any of the methods and compositions for usedisclosed herein, a non-responder to a CAR19 therapy has, or isidentified as having, the biomarker comprising one or more of PD-1+immune effector cells, TIM-3+ immune effector cells, LAG-3+ immuneeffector cells, KLRG1+ immune effector cells, CD27-immune effectorcells, activated T_(EFF) cells, activated T_(REG) cells, activated TH1,activated TH2 cells, stimulated memory cells, or late T memory cells, ora combination thereof,

In some embodiments of any of the methods and compositions for usedisclosed herein, a non-responder to a CAR19 therapy has, or isidentified as having, the biomarker profile of Table 10.

In some embodiments of any of the methods and compositions for usedisclosed herein, expression of one, two, three, four or more (all) ofKLRG1, CD57, CD27, CD122, or CD62L is predictive of patient response toCTL019 therapy.

In some embodiments of any of the methods and compositions for usedisclosed herein, a non-relapser is a patient with B-ALL, and has, or isidentified as having, one or more expression profiles (e.g., protein orgene expression profiles) or gene signatures characteristic of restingT_(EFF) cells or resting T_(REG) cells.

In some embodiments of any of the methods and compositions for usedisclosed herein, a relapser is a patient with B-ALL, and has, or isidentified as having, one or more expression profiles (e.g., protein orgene expression profiles) or gene signatures characteristic of activatedT_(EFF) cells or activated T_(REG) cells.

In some embodiments of any of the aforesaid methods and compositions foruse, a T_(REG) cell (e.g., an activated T_(REG) cell) has upregulatedexpression of one or more (e.g., at least 10, 20, 30, 40, 50, 60, 70, orall) of the following biomarkers: AIM2, ALAS1, BATF, C5orf32, CCL17,CD40LG, CHAC2, CSF1, CTSL1, EBNA1BP2, EDARADD, EMP1, EPAS1, FABP5,FAM40B, FKBP4, FOSL1, GCLM, GK, GPR56, HMOX1, HSPD1, HSPE1, IKBIP, IL10,IL13, IL15RA, IL1RN, IL2RA, IL3, IL4, IL5, IL9, KCNK5, LTA, MANF,MIR1182, MIR155, MIR155HG, MYOF, NDUFAF1, NLN, NME1, NME1-NME2, PANX2,PDIA6, PGAM4, PPIL1, PPPDE2, PRDX4, PRKAR1B, PSMD1, PSMD11, PUS7, RBBP8,SLC27A2, SLC39A14, SLC43A3, SRXN1, STIP1, STT3A, TBX21, TNFRSF11A,TNFRSF1B, TNFRSF8, TNFRSF9, TXN, UCK2, VDR, VTRNA1-3, WDR12, YWHAG,ZDHHC16, or ZNF282. The upregulated expression may be, e.g., measured 16hours after stimulation. The upregulated expression may be determined,e.g., by measuring RNA levels for the indicated genes.

In some embodiments of any of the aforesaid methods and compositions foruse, a T_(EFF) cell (e.g., an activated T_(EFF) cell) has upregulatedexpression of one or more (e.g., at least 10, 20, 30, 40, 50, 60, 70, orall) of the following biomarkers: AIM2, ALAS1, B4GALT5, BATF, C3orf26,C4orf43, CCL3, CCL4, CCT3, CCT7, CD40LG, CHAC2, CSF2, CTNNA1, EBNA1BP2,EDARADD, EEF1E1, EIF2B3, EIF2S1, FABP5, FAM40B, FKBP4, FOSL1, GFOD1,GLRX2, HSPD1, HSPE1, IFNG, IL15RA, IL21, IL2RA, IL3, KCNK5, KIAA0020,LARP4, LRP8, LTA, MANF, MIR1182, MIR155, MIR155HG, MTCH2, MYOF, NDUFAF1,NLN, NME1, NME1-NME2, OTUD7B, PAM, PDIA6, PEA15, PFKM, PGAM1, PGAM4,PPIL1, PRDX4, PRSS23, PSMD1, PSMD11, PSMD14, PTRH2, PUS7, RBBP8, RPF2,RPP25, SFXN1, SLC27A2, SLC39A14, SLC43A3, SORD, SPR, SRXN1, STIP1,STT3A, TBX21, TMCC2, TMEM165, TNFRSF9, TXN, TXNDC5, UCK2, VDR, WDR12,YWHAG, or ZDHHC16. The upregulated expression may be, e.g., measured 16hours after stimulation. The upregulated expression may be determined,e.g., by measuring RNA levels for the indicated genes.

In some embodiments of any of the methods and compositions for usedisclosed herein, a relapser is a patient with B-ALL and has, or isidentified as having, one or more protein or gene expression profilescomprising one, two, three, four, five, ten or more genes according toTable 7A, Table 7B or FIG. 2B or a combination thereof.

In some embodiments of any of the methods and compositions for usedisclosed herein, a relapser has, or is identified as having, anelevated level of one or more T_(REG) cell biomarkers, or a combinationthereof. In some embodiments, the relapser has, or is identified ashaving, upregulated expression of one or more (e.g., at least 10, 20,30, 40, 50, 60, 70, or all) of the following genes: AIM2, ALAS1, BATF,C5orf32, CCL17, CD40LG, CHAC2, CSF1, CTSL1, EBNA1BP2, EDARADD, EMP1,EPAS1, FABP5, FAM40B, FKBP4, FOSL1, GCLM, GK, GPR56, HMOX1, HSPD1,HSPE1, IKBIP, IL10, IL13, IL15RA, IL1RN, IL2RA, IL3, IL4, IL5, IL9,KCNK5, LTA, MANF, MIR1182, MIR155, MIR155HG, MYOF, NDUFAF1, NLN, NME1,NME1-NME2, PANX2, PDIA6, PGAM4, PPIL1, PPPDE2, PRDX4, PRKAR1B, PSMD1,PSMD11, PUS7, RBBP8, SLC27A2, SLC39A14, SLC43A3, SRXN1, STIP1, STT3A,TBX21, TNFRSF11A, TNFRSF1B, TNFRSF8, TNFRSF9, TXN, UCK2, VDR, VTRNA1-3,WDR12, YWHAG, ZDHHC16, or ZNF282. In certain embodiment, the relapserhas, or is identified as having, upregulated expression of one or more(e.g., at least 10, 20, 25, or all) of the following genes: C5orf32,CCL17, CSF1, CTSL1, EMP1, EPAS1, GCLM, GK, GPR56, HMOX1, IKBIP, IL10,IL13, IL1RN, IL4, IL5, IL9, MIR155, PANX2, PGAM4, PRKAR1B, TNFRSF11A,TNFRSF1B, TNFRSF8, VTRNA1-3, or ZNF282. The upregulated expression maybe, e.g., measured 16 hours after stimulation. The upregulatedexpression may be determined, e.g., by measuring RNA levels for theindicated genes.

In some embodiments of any of the methods and compositions for usedisclosed herein, a subject has, or is identified as having, an elevatedlevel of one or more T_(EFF) cell biomarkers, or a combination thereof.In some embodiments, the subject has, or is identified as having,upregulated expression of one or more (e.g., at least 10, 20, 30, 40,50, 60, 70, or all) of the following genes: AIM2, ALAS1, B4GALT5, BATF,C3orf26, C4orf43, CCL3, CCL4, CCT3, CCT7, CD40LG, CHAC2, CSF2, CTNNA1,EBNA1BP2, EDARADD, EEF1E1, EIF2B3, EIF2S1, FABP5, FAM40B, FKBP4, FOSL1,GFOD1, GLRX2, HSPD1, HSPE1, IFNG, IL15RA, IL21, IL2RA, IL3, KCNK5,KIAA0020, LARP4, LRP8, LTA, MANF, MIR1182, MIR155, MIR155HG, MTCH2,MYOF, NDUFAF1, NLN, NME1, NME1-NME2, OTUD7B, PAM, PDIA6, PEA15, PFKM,PGAM1, PGAM4, PPIL1, PRDX4, PRSS23, PSMD1, PSMD11, PSMD14, PTRH2, PUS7,RBBP8, RPF2, RPP25, SFXN1, SLC27A2, SLC39A14, SLC43A3, SORD, SPR, SRXN1,STIP1, STT3A, TBX21, TMCC2, TMEM165, TNFRSF9, TXN, TXNDC5, UCK2, VDR,WDR12, YWHAG, or ZDHHC16. In certain embodiment, the subject has, or isidentified as having, upregulated expression of one or more (e.g., atleast 10, 20, 25, or all) of the following genes: B4GALT5, C3orf26,C4orf43, CCL3, CCL4, CCT3, CCT7, CSF2, CTNNA1, EEF1E1, EIF2B3, EIF2S1,GFOD1, GLRX2, IL21, IL2RA, IL3, KIAA0020, LARP4, LRP8, OTUD7B, PAM,PEA15, PFKM, PGAM1, PGAM4, PRSS23, PSMD1, PSMD11, PSMD14, PTRH2, RPF2,SORD, SPR, TMCC2, TMEM165, or TXNDC5. The upregulated expression may be,e.g., measured 16 hours after stimulation. The upregulated expressionmay be determined, e.g., by measuring RNA levels for the indicatedgenes.

In some embodiments of any of the methods and compositions for usedisclosed herein, the responder (e.g., the complete or partialresponder) has one, two, three or more (or all) of the followingprofile:

(i) has a greater number of CD27+ immune effector cells compared to areference value, e.g., a non-responder number of CD27+ immune effectorcells;

(ii) has a greater number of CD8+ T cells compared to a reference value,e.g., a non-responder number of CD8+ T cells;

(iii) has a lower number of immune cells expressing one or morecheckpoint inhibitors, e.g., a checkpoint inhibitor chosen from PD-1,LAG-3, TIM-3, or KLRG-1, or a combination, compared to a referencevalue, e.g., a non-responder number of cells expressing one or morecheckpoint inhibitors; or

(iv) has a greater number of one, two, three, four or more (all) ofresting T_(EFF) cells, resting T_(REG) cells, younger cells, naïve CD4cells, unstimulated memory cells or early memory T cells, or acombination thereof, compared to a reference value, e.g., anon-responder number of resting T_(EFF) cells, resting T_(REG) cells,naïve CD4 cells, unstimulated memory cells or early memory T cells.

In some embodiments of any of the methods and compositions for usedisclosed herein, the cytokine level or activity, e.g., of (vi), ischosen from one, two, three, four, five, six, seven, eight, or more (orall) of cytokine CCL20/MIP3a, IL17A, IL6, GM-CSF, IFNγ, IL10, IL13, IL2,IL21, IL4, IL5, IL9 or TNFα, or a combination thereof. The cytokine canbe chosen from one, two, three, four or more (all) of IL-17a, CCL20,IL2, IL6, or TNFa. In one embodiment, an increased level or activity ofa cytokine is chosen from one or both of IL-17a and CCL20, is indicativeof increased responsiveness or decreased relapse. In some embodiments,the cytokine level is measured after T cell activation.

In some embodiments of any of the methods and compositions for usedisclosed herein, a transduction efficiency of 15% or higher, e.g., in(vii), is indicative of increased responsiveness or decreased relapse.

In some embodiments of any of the methods and compositions for usedisclosed herein, a transduction efficiency of less than 15%, e.g., in(vii), is indicative of decreased responsiveness or increased relapse.

In another aspect, the invention features a method to identify a likelyresponder (e.g., a complete responder or a partial responder, anon-relapser) to a therapy comprising a CAR-expressing cell (e.g., a Tcell, an NK cell) (e.g., a CD19 CAR-expressing cell therapy, e.g.,described herein, e.g., a CTL019 therapy). In an embodiment, a responderstatus (e.g. a complete responder, a partial responder, a non-responder,a relapser or a non-relapser to a therapy comprising a CAR-expressingcell (e.g., a T cell, an NK cell)) is determined by measuring one ormore of a CD19 CAR-expressing cell gene set signature, a biomarkerlisted in Table 1A, Table 1B, Table 7A, Table 7B, Table 8, Table 9,Table 10, Table 14, Table 15, Table 16 (e.g., CCL20, IL-17a and/orIL-6), Table 17, Table 18, Table 20, a CD27 biomarker, a CD45RObiomarker, a PD-1 biomarker, a LAG-3 biomarker, a TIM-3 biomarker, anIL2RA biomarker, an IL21 biomarker, a CD4 biomarker, a CD8 biomarker, aTH1+ helper T cell gene set signature, a TH2+ helper T cell gene setsignature, a memory T cell (e.g., a CD8+ memory T cell, e.g., a naïve Tcell (T_(N)), e.g. a memory stem cell (T_(SCM)), e.g. a central memory Tcell (T_(CM)), e.g. an effector memory T cell (T_(EM))) gene setsignature, and combinations thereof. In one embodiment, a completeresponder (CR) has, e.g., two, three, four or more (e.g., all) of CD27⁺,CD45RO⁻, PD-1⁻, LAG-3⁻, and TIM-3⁻, as described in Table 9. In oneembodiment, a non-responder (NR) has, e.g., two, three, or more of(e.g., all) of CD27⁻ CD45RO⁺, PD-1⁺, LAG-3⁺, and TIM-3⁺, as described inTable 10.

In an embodiment, the responder or relapser status (e.g. completeresponder, partial responder, non-responder, relapser or non-relapser toa CAR-expressing cell therapy) is determined by evaluating, e.g.,measuring, two, three, four, five, six, seven, eight, nine, ten, fifteenor more of a CD19 CAR-expressing cell gene set signature, a biomarkerlisted in Table 1A, Table 1B, Table 7A, Table 7B, Table 8, Table 9,Table 10, Table 14, Table 15, Table 16 (e.g., CCL20, IL-17a and/orIL-6), Table 17, Table 18, Table 20, PD-1, LAG-3, TIM-3, CD57, CD27,CD122, CD62L, and KLRG1.

In an embodiment, any of the methods and compositions for use disclosedherein can be used prior to administration of a CAR-expressing celltherapy. In some embodiments, provided methods can be used before, atthe same time, or during course of a CAR-expressing cell therapy.

In an embodiment, any of the methods and compositions for use disclosedherein can be used to identify a subject having cancer, e.g., ahematological cancer such as, e.g., CLL or ALL, as having an increasedor a decreased likelihood to respond to a treatment that comprises aCAR-expressing cell (e.g., T cell, NK cell) therapy, e.g., a CD19CAR-expressing cell therapy. The method comprises: (1) acquiring asample from the subject (e.g., an apheresis sample obtained from theblood of the subject; and/or e.g., a manufactured product sample, e.g.,genetically engineered T cells); (2) determining a level (e.g., amountor activity) of one or more biomarkers (e.g., 2, 3, 4, 5, 10, 15 ormore) listed in Table 1A, Table 1B, Table 7A, Table 7B, Table 8, Table9, Table 10, Table 14, Table 15, Table 16 (e.g., CCL20, IL-17a and/orIL-6), Table 17, Table 18, Table 20, PD-1, LAG-3, TIM-3, CD57, CD27,CD122, CD62L, and KLRG1 in the sample; and (3) (optionally) comparingthe determined level of the one or more markers to a reference level;and (4) identifying the subject as a complete responder, partialresponder, non-responder, a relapser or non-relapser to theCAR-expressing cell therapy. In embodiments, a difference, e.g., astatistically significant difference, between the determined levelcompared to a reference level is predictive of the subjectsresponsiveness to the CAR-expressing cell therapy.

In an aspect, provided methods comprise (1) acquiring a sample (e.g., anapheresis sample obtained from the subject; and/or e.g., a manufacturedproduct sample, e.g., genetically engineered T cells, e.g., amanufactured CD19 CAR-expressing cell product); (2) acquiring, e.g.,determining a gene signature of the sample; and (3) (optionally)comparing the gene signature to a reference gene signature; wherein adifference, e.g., a statistically significant difference, in expressionlevel of one or more of the biomarkers in the determined gene signatureis predictive of the subjects responsiveness to the CAR-expressing celltherapy. In an embodiment, the gene signature comprises one or moremarkers selected from Table 1A, Table 1B, Table 2, Table 3, Table 4,Table 5, Table 6, Table 7A, Table 7B, Table 8, Table 9, Table 10, Table14 (e.g., CCL20, IL-17a and/or IL-6), Table 17, Table 18, Table 20,PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L, KLRG1, and combinationsthereof. In one embodiment, the sample is a biological sample selectedfrom a blood, plasma, or a serum sample. In a particular embodiment, abiological sample is a blood sample. In an embodiment, the sample is anapheresis sample, e.g., immune effector cells (e.g., T cells) obtainedfrom the blood of the subject. In an embodiment, the sample is amanufactured product sample, e.g. genetically engineered T cells, e.g.,a manufactured CD19 CAR-expressing cell product.

In an aspect, methods are provided for determining the responsiveness ofa subject having cancer, e.g., a hematological cancer such as, e.g., CLLor ALL, to a treatment comprising a CAR-expressing cell (e.g., a T cell,an NK cell) therapy, e.g., a CD19 CAR-expressing cell therapy, e.g.,described herein. The method includes: determining a level or activityof one or more biomarkers (e.g., 2, 3, 4, 5, 10, 15 or more) listed inTable 1A, Table 1B, Table 7A, Table 7B, Table 8, Table 9, Table 10,Table 14, Table 15, Table 16 (e.g., CCL20, IL-17a and/or IL-6), Table17, Table 18, Table 20, PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L,and KLRG1 in a sample obtained prior to treatment; wherein a difference,e.g., a statistically significant difference, in a level (e.g., amountor activity) of one or more markers in the sample relative to apredetermined value is indicative of increased responsiveness to theCAR-expressing cell. In one embodiment, the sample is a biologicalsample selected from a blood, plasma, or a serum sample. In a particularembodiment, a biological sample is a blood sample. In one embodiment,the sample is an apheresis sample, e.g., T cells obtained from the bloodof the subject. In an embodiment, the sample is a manufactured productsample, e.g. genetically engineered T cells, e.g., obtained from theblood of the subject, e.g., a manufactured CAR-expressing cell product,e.g., a manufactured CD19 CAR-expressing cell product.

In an embodiment, methods are provided for evaluating a subject havingcancer, e.g., a hematological cancer such as, e.g., CLL or ALL,comprising acquiring a value of responder or relapser status for thesubject that comprises a measure of one or more (e.g., 2, 3, 4, 5, 10,15 or more) of the following: a biomarker listed in Table 1A, Table 1B,Table 7A, Table 7B, Table 8, Table 9, Table 10, Table 14, Table 15,Table 16 (e.g., CCL20, IL-17a and/or IL-6), Table 17, Table 18, Table20, a CD27 biomarker, a CD45RO biomarker, a PD-1 biomarker, a LAG-3biomarker, a TIM-3 biomarker, an IL2RA biomarker, an IL21 biomarker, aCD4+ biomarker, a CD8+ biomarker, a TH1+ helper T cell gene setsignature, a TH2+ helper T cell gene set signature, a memory T cell(e.g., a CD8+ memory T cell, e.g., a naïve T cell (T_(N)), e.g. a memorystem cell (T_(SCM)), e.g. a central memory T cell (T_(CM)), e.g. aneffector memory T cell (T_(EM))) gene set signature, and a CD19CAR-expressing cell (e.g., T cell, NK cell) gene set signature, therebyevaluating the subject. In an embodiment, methods comprise a measure ofone or more of the following: a biomarker listed in Table 1A, Table 1B,Table 7A, Table 7B, Table 8, Table 9, Table 10, Table 14, Table 15,Table 16 (e.g., CCL20, IL-17a and/or IL-6), Table 17, Table 18, Table20, PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L, KLRG1, and a CD19CAR-expressing cell gene set signature. In one embodiment, methodscomprise a measure of one or more of the following: a biomarker listedin Table 1A, Table 1B, Table 7A, Table 7B, Table 8, Table 9, Table 10,Table 14, Table 15, Table 16 (e.g., CCL20, IL-17a and/or IL-6), Table17, Table 18, Table 20, PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L,KLRG1, and a CD19 CAR-expressing cell gene set signature, for evaluatinga subject having CLL. In another embodiment, methods comprise a measureof one or more of the following: a biomarker listed in Table 1A, Table1B, Table 7A, Table 7B, Table 8, Table 9, Table 10, Table 14, Table 15,Table 16 (e.g., CCL20, IL-17a and/or IL-6), Table 17, Table 18, Table20, PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L, KLRG1, and a CD19CAR-expressing cell gene set signature, for evaluating a subject havingALL. In one embodiment, the method comprises a measure of one or more(e.g., 2, 3, 4, 5, 10, 15 or more) of the following: a biomarker listedin Table 1A, Table 1B, Table 7A, Table 7B, Table 8, Table 9, Table 10,Table 14, Table 15, Table 16 (e.g., CCL20, IL-17a and/or IL-6), Table17, Table 18, Table 20, PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L,KLRG1, or a CD19 CAR-expressing cell gene set signature in a biologicalsample selected from a blood, plasma, or a serum sample. In a particularembodiment, a biological sample is a blood sample. In an embodiment, thesample is an apheresis sample, e.g., T cells obtained from the blood ofthe subject. In an embodiment, the sample is a manufactured productsample, e.g. genetically engineered T cells, e.g., obtained from theblood of the subject, e.g., a manufactured CAR-expressing cell product,e.g., a manufactured CD19 CAR-expressing cell product.

In an embodiment, methods are provided for evaluating or monitoring theeffectiveness of a CAR-expressing cell (e.g., T cell, NK cell) therapy,e.g., a CD19 CAR-expressing cell therapy, in a subject having cancercomprising acquiring a value of responder or relapser status for thesubject that comprises a measure of one or more (e.g., 2, 3, 4, 5, 10,15 or more) of the following: a biomarker listed in Table 1A, Table 1B,Table 7A, Table 7B, Table 8, Table 9, Table 10, Table 14, Table 15,Table 16 (e.g., CCL20, IL-17a and/or IL-6), Table 17, Table 18, Table20, a CD27 biomarker, a CD45RO biomarker, a PD-1 biomarker, a LAG-3biomarker, a TIM-3 biomarker, an IL2RA biomarker, an IL21 biomarker, aCD4 biomarker, a CD8 biomarker, a TH1+ helper T cell gene set signature,a TH2+ helper T cell gene set signature, a memory T cell (e.g., a CD8+memory T cell, e.g., a naïve T cell (T_(N)), e.g. a memory stem cell(T_(SCM)), e.g. a central memory T cell (T_(C)M), e.g. an effectormemory T cell (T_(EM))) gene set signature, and a CD19 CAR-expressingcell gene set signature, thereby evaluating or monitoring theeffectiveness of the CAR-expressing cell therapy in the subject. In anembodiment, methods comprise a measure of one or more (e.g., 2, 3, 4, 5,10, 15 or more) of the following: a biomarker listed in Table 1A, Table1B, Table 7A, Table 7B, Table 8, Table 9, Table 10, Table 14, Table 15,Table 16 (e.g., CCL20, IL-17a and/or IL-6), Table 17, Table 18, Table20, PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L, KLRG1, or a CD19CAR-expressing cell gene set signature. In an embodiment, methodscomprise a measure of one or more (e.g., 2, 3, 4, 5, 10, 15 or more) ofthe following: a biomarker listed in Table 1A, Table 1B, Table 7A, Table7B, Table 8, Table 9, Table 10, Table 14, Table 15, Table 16 (e.g.,CCL20, IL-17a and/or IL-6), Table 17, Table 18, Table 20, PD-1, LAG-3,TIM-3, CD57, CD27, CD122, CD62L, KLRG1, or a CD19 CAR-expressing cellgene set signature, for evaluating or monitoring the effectiveness of aCAR-expressing cell therapy in a subject having CLL. In anotherembodiment, methods comprise a measure of one or more (e.g., 2, 3, 4, 5,10, 15 or more) of the following: a biomarker listed in Table 1A, Table1B, Table 7A, Table 7B, Table 8, Table 9, Table 10, Table 14, Table 15,Table 16 (e.g., CCL20, IL-17a and/or IL-6), Table 17, Table 18, Table20, PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L, KLRG1, or a CD19CAR-expressing cell gene set signature, for evaluating or monitoring theeffectiveness of a CAR-expressing cell therapy in a subject having ALL.In one embodiment, the method comprises a measure of one or more (e.g.,2, 3, 4, 5, 10, 15 or more) of the following: a biomarker listed inTable 1A, Table 1B, Table 7A, Table 7B, Table 8, Table 9, Table 10,Table 14, Table 15, Table 16 (e.g., CCL20, IL-17a and/or IL-6), Table17, Table 18, Table 20, PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L,KLRG1, or a CD19 CAR-expressing cell gene set signature in a biologicalsample selected from a blood, plasma, or a serum sample. In oneembodiment, a biological sample is a blood sample. In an embodiment, thesample is an apheresis sample, e.g., T cells obtained from the blood ofthe subject. In an embodiment, the sample is a manufactured productsample, e.g. genetically engineered T cells, e.g., obtained from theblood of the subject.

In an embodiment, methods are provided for providing a prediction forsuccess rate of a CAR-expressing cell therapy, e.g., a CD19CAR-expressing cell therapy, e.g., described herein, in a subject havingcancer, said method comprising steps of providing a biological samplefrom the subject; determining the levels of expression of one or more(e.g., 2, 3, 4, 5, 10, 15 or more) genes listed in Table 1A, Table 1B,Table 7A, Table 7B, Table 8, Table 9, Table 10, Table 14, Table 15,Table 16 (e.g., CCL20, IL-17a and/or IL-6), and Table 17 to obtain agene expression pattern for the sample; and based on the gene expressionpattern obtained, providing a prognosis to the subject. In anembodiment, a biological sample includes, but is not limited to a blood,plasma, or a serum sample. In a particular embodiment, a biologicalsample is a blood sample. In one embodiment, the sample is an apheresissample, e.g., T cells obtained from the blood of the subject. In anembodiment, the subject has CLL. In an embodiment, the subject has ALL.

In another aspect, methods for treating a subject having cancer, e.g., ahematological cancer, are provided. In an embodiment, methods areprovided for treating a subject having cancer determining if a subjecthas a difference, e.g., a statistically significant difference, inexpression level of one or more (e.g., 2, 3, 4, 5, 10, 15 or more)markers listed in Table 1A, Table 1B, Table 7A, Table 7B, Table 8, Table9, Table 10, Table 14, Table 15, Table 16 (e.g., CCL20, IL-17a and/orIL-6) and Table 17 relative to a reference level, and if there is adifference, e.g., a statistically significant difference, between thedetermined level and reference level, administering to the subject atherapeutically effective dose of a CAR-expressing cell (e.g., T cell,NK cell), thereby treating the subject. In an embodiment, wherein thereis a difference, e.g., a statistically significant difference, betweenthe determined level and reference level, the method comprises modifyingthe CAR-expressing cell product prior to infusion into the subject. Inan embodiment, wherein there is a difference, e.g., a statisticallysignificant difference, between the determined level and the referencelevel, the method comprises modifying the manufacture of aCAR-expressing cell product prior to infusion into the subject. In anembodiment, if there is a difference, e.g., a statistically significantdifference, between the determined level and reference level, the methodcomprises adjusting the CAR-expressing cell infusion dose to achieve ananti-cancer effect.

In an embodiment, the methods of treatment described herein comprisedetermining if a subject has an increased likelihood to respond to aCAR-expressing cell (e.g., T cell, NK cell) therapy, e.g., a CD19CAR-expressing cell therapy, e.g., a CD19 CAR-expressing cell therapydescribed herein, by comparing the level of one or more (e.g., 2, 3, 4,5, 10, 15 or more) markers in Table 1A, Table 1B, Table 7A, Table 7B,Table 8, Table 9, Table 10, Table 14, Table 15 and Table 16 (e.g.,CCL20, IL-17a and/or IL-6) in a sample from the subject relative to areference level, wherein a difference, e.g., a statistically significantdifference, in expression level of one or more maker genes relative tothe reference level is indicative of an increased likelihood ofresponse; and administering to the subject a therapeutically effectivedose of a CAR-expressing cell, thereby treating the subject. In oneembodiment, the sample is selected from a blood, plasma or a serumsample. In one embodiment, the sample is an apheresis sample, e.g., Tcells obtained from the blood of the subject.

In an embodiment, the methods of treatment described herein furthercomprise obtaining a sample from a subject; determining a level of oneor more (e.g., 2, 3, 4, 5, 10, 15 or more) markers in Table 1A, Table1B, Table 7A, Table 7B, Table 8, Table 9, Table 10, Table 14, Table 15,Table 16 (e.g., CCL20, IL-17a and/or IL-6), Table 17, Table 18, Table20, PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L, and KLRG1, in thesample; comparing the determined level of one or more markers in Table1A, Table 1B, Table 7A, Table 7B, Table 8, Table 9, Table 10, Table 14,Table 15, Table 16 (e.g., CCL20, IL-17a and/or IL-6), Table 17, Table18, Table 20, PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L, and KLRG1,to a reference level; and administering a therapeutically effective doseof a CAR-expressing cell (e.g., T cell, NK cell), if the subject isidentified as having a difference, e.g., a statistically significantdifference, in the determined level of one or more markers in Table 1A,Table 1B, Table 7A, Table 7B, Table 8, Table 9, Table 10, Table 14,Table 15, Table 16 (e.g., CCL20, IL-17a and/or IL-6), Table 17, Table18, Table 20, PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L, and KLRG1,to a reference level in the sample.

In an embodiment, the methods of treatment described herein comprise, orfurther comprise, acquiring a value of responder or relapser status forthe subject that comprises a measure of one or more (e.g., 2, 3, 4, 5,10, 15 or more) of the following: a biomarker listed in Table 1A, Table1B, Table 7A, Table 7B, Table 8, Table 9, Table 10, Table 14, Table 15,Table 16 (e.g., CCL20, IL-17a and/or IL-6), Table 17, Table 18, Table20, a CD27 biomarker, a CD45RO biomarker, a PD-1 biomarker, a LAG-3biomarker, a TIM-3 biomarker, an IL2RA biomarker, an IL21 biomarker, aCD4 biomarker, a CD8 biomarker, a TH1+ helper T cell gene set signature,a TH2+ helper T cell gene set signature, a memory T cell (e.g., a CD8+memory T cell, e.g., a naïve T cell (T_(N)), e.g. a memory stem cell(T_(SCM)), e.g. a central memory T cell (T_(CM)), e.g. an effectormemory T cell (T_(EM))) gene set signature, and a CD19 CAR-expressingcell gene set signature, and responsive to a determination of responderor relapser status, performing one, two, three four or more of:identifying the subject as a complete responder, partial responder ornon-responder; administering a CAR-expressing cell therapy; selecting oraltering a dosing of a CAR-expressing cell therapy; selecting oraltering the schedule or time course of a CAR-expressing cell therapy;administering, e.g., to a non-responder or a partial responder, anadditional agent in combination with a CAR-expressing cell therapy,e.g., a checkpoint inhibitor, e.g., a checkpoint inhibitor describedherein, or a kinase inhibitor, e.g., a kinase inhibitor describedherein; administering to a non-responder or partial responder a therapythat increases the number of naïve T cells in the subject prior totreatment with a CAR-expressing cell therapy; modifying a manufacturingprocess of a CAR-expressing cell therapy, e.g., enrich for naïve T cellsprior to introducing a nucleic acid encoding a CAR, e.g., for a subjectidentified as a non-responder or a partial responder; or selecting analternative therapy, e.g., a standard of care for a particular cancer(e.g., as described herein), e.g., for a non-responder, partialresponder or relapser; thereby treating cancer in the subject.

Systems

In still another aspect, the present disclosure provides kits forpredicting subject response to CAR-expressing cell (e.g., T cell, NKcell) therapy, e.g., a CD19 CAR-expressing cell therapy, e.g., a CD19CAR-expressing cell therapy described herein. The kits comprise at leastone reagent that specifically detects the level or activity of a set ofgenes (e.g., 2, 3, 4, 5, 10, 15 or more of the genes) selected fromTable 1A, Table 1B, Table 7A, Table 7B, Table 8, Table 9, Table 10,Table 14, Table 15, Table 16 (e.g., CCL20, IL-17a and/or IL-6), Table17, Table 18, Table 20, PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L,and/or KLRG1; and instructions for using the kits, e.g., for predictinga subject's response to a CAR-expressing cell therapy. In someembodiments, said instructions for use provide that if one or more ofthe detected expression levels is different from, e.g., greater than areference level, the subject is more likely to respond positively to aCAR-expressing cell therapy. In some embodiments, said instructions foruse provide that if one or more of the detected expression levels isless than a reference level, the subject is more likely to respondpositively to a CAR-expressing cell therapy. In some embodiments, if thelevel or activity of PD-1, LAG-3, or TIM-3, or any combination thereof,is less than a reference value, the subject is more likely to respondpositively to the therapy.

In certain embodiments, at least one reagent that specifically detectsexpression levels of the set of genes comprises a nucleic acid probecomplementary to mRNA expressed from the genes, for example a CDNA or anoligonucleotide. The nucleic acid probe may be immobilized on asubstrate surface or may be in solution. The set of reagents may detectthe expression of polypeptides, e.g., surface polypeptides, encoded bysaid set of genes. In one embodiment, the nucleic acid probe comprises anucleic acid of about 10, 15, 20, 25, 30, 35, 40, 45, 50 or 100 nucleicacid residues complementary the nucleic acid sequence of a biomarkerlisted in Table 1A, Table 1B, Table 7A, Table 7B, Table 8, Table 9,Table 10, Table 14, Table 15, Table 16 (e.g., CCL20, IL-17a and/orIL-6), Table 17, Table 18, Table 20, PD-1, LAG-3, TIM-3, CD57, CD27,CD122, CD62L, and/or KLRG1. The kits may further comprise one or moreof: extraction buffer/reagents and protocol, amplificationbuffer/reagents and protocol, hybridization buffer/reagents andprotocol, and labeling buffer/reagents and protocol.

In certain embodiments, the kits further comprise at least one CD19CAR-expressing cell (e.g., T cell, NK cell) gene set signature. In oneembodiment, the kit further comprises a reference standard.

In an embodiment, the subject has CLL.

In an embodiment, the subject has ALL.

In an embodiment, the subject has B-cell ALL.

In an aspect, the disclosure features a reaction mixture comprising atleast one reagent that specifically detects expression levels of a setof genes (e.g., 2, 3, 4, 5, 10, 15 or more of the genes) selected fromTable 1A, Table 1B, Table 7A, Table 7B, Table 8, Table 9, Table 10,Table 14, Table 15, Table 16 (e.g., CCL20, IL-17a and/or IL-6), Table17, Table 18, Table 20, PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L,and/or KLRG1; and a biological sample. In one embodiment, the sample isselected from a blood, plasma or a serum sample. In one embodiment, thesample is an apheresis sample, e.g., T cells obtained from the blood ofthe subject. In one embodiment, the sample comprises CAR-expressingcells, e.g., CART-expressing cells, e.g., CART19 cells.

In certain embodiments, at least one reagent that specifically detectsexpression levels of the set of genes comprises a nucleic acid probecomplementary to mRNA expressed from the genes, for example a CDNA or anoligonucleotide. The nucleic acid probe may be immobilized on asubstrate surface or may be in solution. The set of reagents may detectthe expression of polypeptides, e.g., surface polypeptides, encoded bysaid set of genes. In one embodiment, the nucleic acid probe comprises anucleic acid of about 10, 15, 20, 25, 30, 35, 40, 45, 50 or 100 nucleicacid residues complementary the nucleic acid sequence of a biomarkerlisted in Table 1A, Table 1B, Table 7A, Table 7B, Table 8, Table 9,Table 10, Table 14, Table 15, Table 16 (e.g., CCL20, IL-17a and/orIL-6), Table 17, Table 18, Table 20, PD-1, LAG-3, TIM-3, CD57, CD27,CD122, CD62L, and/or KLRG1. The reaction mixture may further compriseone or more of: extraction buffer/reagents, amplificationbuffer/reagents, hybridization buffer/reagents, and labelingbuffer/reagents.

In another aspect, the present disclosure features a system forevaluating cancer in a subject. The system comprises at least oneprocessor operatively connected to a memory, the at least one processorwhen executing is configured to perform any one or more of the stepsdescribed herein.

In still another aspect, the present disclosure features a system forevaluating cancer in a subject. The system includes at least oneprocessor operatively connected to a memory, the at least one processorwhen executing is configured to:

-   -   acquire a value of responder or relapser status that comprises a        measure of one, two, three, four, five, six, seven or more (all)        of the following:

(i) the level or activity of CD27 and/or CD45RO− (e.g., CD27+CD45RO−)immune effector cells, e.g., in a CD4+ or a CD8+ T cell population in asample (e.g., an apheresis sample or a manufactured CAR-expressing cellproduct sample);

(ii) the level or activity of one, two, three, or more (e.g., all) ofresting T_(EFF) cells, resting T_(REG) cells, younger T cells (e.g.,younger CD4 or CD8 cells, or gamma/delta T cells), early memory T cells,or a combination thereof, in a sample (e.g., an apheresis sample or amanufactured CAR-expressing cell product sample);

(iii) the level or activity of one, two, three, or more (e.g., all) ofactivated T_(EFF) cells, activated T_(REG) cells, older T cells (e.g.,older CD4 or CD8 cells), or late memory T cells, or a combinationthereof, in a sample (e.g., an apheresis sample or a manufacturedCAR-expressing cell product sample);

(iv) the level or activity of an immune cell exhaustion marker, e.g.,one, two or more immune checkpoint inhibitors (e.g., PD-1, TIM-3 and/orLAG-3) in a sample (e.g., an apheresis sample or a manufacturedCAR-expressing cell product sample);

(v) the level or activity of one, two, three, four, five, ten, twenty ormore of the biomarkers listed in Table 1A, Table 1B, Table 7A, Table 7B,Table 8, Table 9, Table 10, Table 14 (e.g., CCL20, IL-17a and/or IL-6),Table 16, Table 17, Table 18, Table 20, FIG. 2B, PD-1, LAG-3, TIM-3,CD57, CD27, CD122, CD62L, KLRG1, or a CD19 CAR-expressing cell gene setsignature;

(vi) a cytokine level or activity (e.g., quality of cytokine repertoire)in a CAR-expressing cell product sample, e.g., CAR19-expressing cellproduct sample (e.g., CTL019), wherein the cytokine is chosen from one,two, three, four, five or more (or all) of the cytokines listed in Table16;

(vii) a transduction efficiency of a CAR-expressing cell in amanufactured CAR-expressing cell product sample; or

(viii) a quantity of CD27+PD-1− cells in a subject, e.g., a sample fromthe subject (e.g., an apheresis sample or a CAR-expressing cell productsample, e.g., CAR19-expressing cell product sample (e.g., CTL019)),e.g., a quantity greater than or equal to 1×10⁷ cells.

responsive to a determination of the value of responder status, performone, two, three, four, five, six, seven, or more (e.g., all) of:

identify the subject as a complete responder, partial responder,non-responder, relapser or non-relapser;

recommend administering a CAR-expressing cell therapy;

recommend a selection or alteration of a dosing of a CAR-expressing celltherapy;

recommend a selection or alteration of a schedule or time course of aCAR-expressing cell therapy;

recommend administering, e.g., to a non-responder or a partialresponder, an additional agent in combination with a CAR-expressing celltherapy, e.g., a checkpoint inhibitor, e.g., a checkpoint inhibitordescribed herein;

recommend administering to a non-responder or partial responder atherapy that increases the number of naïve T cells in the subject priorto treatment with a CAR-expressing cell therapy;

recommend modifying a manufacturing process of a CAR-expressing celltherapy, e.g., enrich for naïve T cells prior to introducing a nucleicacid encoding a CAR, e.g., for a subject identified as a non-responderor a partial responder;

recommend modifying the CAR-expressing cell product prior to infusioninto the patient;

recommend adjusting the CAR-expressing cell infusion dose to achieveclinical efficacy;

recommend administering an alternative therapy, e.g., for anon-responder or partial responder or relapser;

recommend a selection of an alternative therapy, e.g., for anon-responder or partial responder, e.g., a standard of care for aparticular cancer type, or if the subject is, or is identified as, anon-responder or a relapser, recommend decreasing the T_(REG) cellpopulation and/or T_(REG) gene signature, e.g., by CD25 depletion,administration of cyclophosphamide, anti-GITR antibody, mTOR inhibitor,or a combination thereof.

In one embodiment, the value includes a measure of a CD19 CAR-expressingcell gene set signature and a combination of one or more (e.g., 2, 3, 4,5, 10, 15 or more) of: a biomarker listed in Table 1A, Table 1B, Table7A, Table 7B, Table 8, Table 9, Table 10, Table 14, Table 15, Table 16(e.g., CCL20, IL-17a and/or IL-6), Table 17, Table 18, Table 20, a CD27biomarker, a CD45RO biomarker, a PD-1 biomarker, a LAG-3 biomarker, aTIM-3 biomarker, an IL2RA biomarker, an IL21 biomarker, a CD4 biomarker,a CD8 biomarker, a TH1+ helper T cell gene set signature, a TH2+ helperT cell gene set signature, and a memory T cell (e.g., a CD8+ memory Tcell, e.g., a naïve T cell (T_(N)), e.g. a memory stem cell (T_(SCM)),e.g. a central memory T cell (T_(CM)), e.g. an effector memory T cell(T_(EM))) gene set signature; and responsive to a determination of thevalue of responder status, perform one, two, three, four or more of:identify the subject as a complete responder, partial responder, ornon-responder; recommend a CAR-expressing cell therapy; recommend aselection or alteration of a dosing of a CAR-expressing cell therapy;recommend an alternative therapy, recommend a combination therapy, e.g.,a combination with a CAR-expressing cell therapy, recommend or alter amanufacturing process of a CAR-expressing cell therapy.

In one embodiment, the at least one processor when executing isconfigured to: acquire a value of responder status that comprises ameasure of a CD19 CAR-expressing cell gene set signature and acombination of one or more (e.g., 2, 3, 4, 5, 10, 15 or more) of: abiomarker listed in Table 1A, Table 1B, Table 7A, Table 7B, Table 8,Table 9, Table 10, Table 14, Table 15, Table 16 (e.g., CCL20, IL-17aand/or IL-6), Table 17, Table 18, Table 20, PD-1, LAG-3, TIM-3, CD57,CD27, CD122, CD62L, and KLRG1; and responsive to a determination of thevalue of responder status, perform one, two, three, four or more of:identify the subject as a complete responder, partial responder, ornon-responder; recommend a CAR-expressing cell therapy; recommend aselection or alteration of a dosing of a CAR-expressing cell therapy;recommend an alternative therapy, e.g., a standard of care for aparticular cancer (e.g., as described herein); recommend a combinationtherapy, e.g., a combination with a CAR-expressing cell therapy,recommend or alter a manufacturing process of a CAR-expressing celltherapy, e.g., as described herein.

Manufacturing Methods

In another aspect, the invention features a method of evaluating thepotency of a CAR-expressing cell product, e.g., CAR19-expressing cellproduct sample (e.g., CTL019). The method includes acquiring a value forone, two, three, four, five, six, seven, eight, or more (e.g., all) of:

(i) the level or activity of CD27 and/or CD45RO− (e.g., CD27+CD45RO−)immune effector cells, e.g., in a CD4+ or a CD8+ T cell population, inthe CAR-expressing cell product;

(ii) the level or activity of one, two, three, or more (e.g., all) ofresting T_(EFF) cells, resting T_(REG) cells, younger T cells (e.g.,younger CD4 or CD8 cells, or gamma/delta T cells), or early memory Tcells, or a combination thereof, in the CAR-expressing cell product;

(iii) the level or activity of one, two, three, or more (e.g., all) ofactivated T_(EFF) cells, activated T_(REG) cells, older T cells (e.g.,older CD4 or CD8 cells), or late memory T cells, or a combinationthereof, in the CAR-expressing cell product;

(iv) the level or activity of an immune cell exhaustion marker, e.g.,one, two or more immune checkpoint inhibitors (e.g., PD-1, TIM-3 and/orLAG-3) in the CAR-expressing cell product;

(v) the level or activity of one, two, three, four, five, ten, twenty ormore of the biomarkers listed in Table 1A, Table 1B, Table 7A, Table 7B,Table 8, Table 9, Table 10, Table 14 (e.g., CCL20, IL-17a and/or IL-6),Table 16, Table 17, Table 18, Table 20, FIG. 2B, PD-1, LAG-3, TIM-3,CD57, CD27, CD122, CD62L, KLRG1, or a CD19 CAR-expressing cell gene setsignature;

(vi) a cytokine level or activity in a CAR-expressing cell productsample, e.g., CAR19-expressing cell product sample (e.g., CTL019),wherein the cytokine is chosen from one, two, three, four, five or more(or all) of the cytokines listed in Table 16;

(vii) a transduction efficiency of CAR-expressing cells in the product;

(viii) a quantity of CD27+PD-1− cells in a subject, e.g., a sample fromthe subject (e.g., an apheresis sample or a CAR-expressing cell productsample, e.g., CAR19-expressing cell product sample (e.g., CTL019)),e.g., a quantity greater than or equal to 1×10⁷ cells; or

(ix) the level or activity of a T_(REG) cell or cell population,

wherein an increase in (i), (ii), (vi), (vii), (viii), or anycombination thereof, is indicative of increased potency of theCAR-expressing cell product, and

wherein an increase in (iii), (iv), (ix), or any combination thereof isindicative of decreased potency of the CAR-expressing cell product.

In a related aspect, the invention features a method for optimizingmanufacturing of a CAR-expressing cell product, e.g., CAR19-expressingcell product sample (e.g., CTL019). The method includes:

(1) acquiring a sample comprising CAR-expressing cell (e.g., apopulation of CAR-expressing immune effector cells);

(2) activating the CAR-expressing cell in vitro;

(3) evaluating the potency of the potency of the activatedCAR-expressing cell by determining one, two, three, four, five, six,seven, eight, or more (e.g., all) of:

(i) the level or activity of CD27 and/or CD45RO− (e.g., CD27+CD45RO−)immune effector cells, e.g., in a CD4+ or a CD8+ T cell population, inthe CAR-expressing cell product;

(ii) the level or activity of one, two, three, or more (e.g., all) ofresting T_(EFF) cells, resting T_(REG) cells, younger T cells (e.g.,younger CD4 or CD8 cells, or gamma/delta T cells), or early memory Tcells, or a combination thereof, in the CAR-expressing cell product;

(iii) the level or activity of one, two, three, or more (e.g., all) ofactivated T_(EFF) cells, activated T_(REG) cells, older T cells (e.g.,older CD4 or CD8 cells), or late memory T cells, or a combinationthereof, in the CAR-expressing cell product;

(iv) the level or activity of an immune cell exhaustion marker, e.g.,one, two or more immune checkpoint inhibitors (e.g., PD-1, TIM-3 and/orLAG-3) in the CAR-expressing cell product;

(v) the level or activity of one, two, three, four, five, ten, twenty ormore of the biomarkers listed in Table 1A, Table 1B, Table 7A, Table 7B,Table 8, Table 9, Table 10, Table 14 (e.g., CCL20, IL-17a and/or IL-6),Table 16, Table 17, Table 18, Table 20, FIG. 2B, PD-1, LAG-3, TIM-3,CD57, CD27, CD122, CD62L, KLRG1, or a CD19 CAR-expressing cell gene setsignature;

(vi) a cytokine level or activity in a CAR-expressing cell productsample, e.g., CAR19-expressing cell product sample (e.g., CTL019),wherein the cytokine is chosen from one, two, three, four, five or more(or all) of the cytokines listed in Table 16;

(vii) a transduction efficiency of CAR-expressing cells in the product;

(viii) a quantity of CD27+PD-1− cells in a subject, e.g., a sample fromthe subject (e.g., an apheresis sample or a CAR-expressing cell productsample, e.g., CAR19-expressing cell product sample (e.g., CTL019)),e.g., a quantity greater than or equal to 1×10⁷ cells; or

(ix) the level or activity of a T_(REG) cell or cell population,

wherein an increase in (i), (ii), (vi), (vii), (viii), or anycombination thereof is indicative of increased potency of theCAR-expressing cell product, and

wherein an increase in (iii), (iv), (ix), or any combination thereof, isindicative of decreased potency of the CAR-expressing cell product.

In a related aspect, disclosed herein is a manufacturing process of aCAR-expressing cell (e.g., T cell, NK cell) product (e.g., a CD19CAR-expressing cell, e.g., a CD19 CAR-expressing cell described herein,e.g., CTL019) to determine the potency or efficacy of the product. In anembodiment, provided methods comprise steps of providing a biologicalsample from a subject, e.g., a blood, serum or plasma sample;determining the levels of expression of one or more (e.g., 2, 3, 4, 5,10, 15 or more) genes listed in Table 1A, 1B, Table 7A, Table 7B, Table8, Table 9, Table 10, Table 14, Table 15, Table 16 (e.g., CCL20, IL-17aand/or IL-6), Table 17, Table 18, Table 20, PD-1, LAG-3, TIM-3, CD57,CD27, CD122, CD62L, and KLRG1, to obtain a gene expression pattern forthe sample; (optionally) comparing the obtained gene expression patternto a predetermined value; and determining a difference between theobtained and the predetermined value. In an embodiment, the determineddifference is recorded in a quality control record.

In some embodiments, any of the methods disclosed herein furthercomprise a step of enriching for, e.g., isolating, cells having anincrease in any of (i), (ii), (vi), (vii), (viii), or any combinationthereof, or a decrease in any of (iii), (iv), (ix), or any combinationthereof.

In another aspect, the invention features a method for manufacturing ofa product sample, e.g., genetically engineered T cells, e.g., obtainedfrom the blood of a subject, e.g., a manufactured CAR-expressing cell(e.g., T cell, NK cell) product, e.g., a CD 19 CAR-expressing cellproduct, e.g., a CD19 CAR-expressing cell product described herein,e.g., CTL019). In an embodiment, the method comprises:

providing a manufactured product sample, e.g., genetically engineered Tcells, e.g., obtained from the blood of a subject, e.g., a manufacturedCAR-expressing cell product, e.g., a CD 19 CAR-expressing cell product,e.g., a CD19 CAR-expressing cell product described herein, e.g.,CTL019);

(i) acquiring a cytokine expression profile (e.g., of Table 14, Table 15or Table 16 (e.g., CCL20, IL-6 and/or IL-17a) secreted from theCAR-expressing cell product; and/or

(ii) acquiring measure of a transduction efficiency of CAR-expressingcells in the product;

identifying the CAR-expressing cell product as suitable foradministration based on the determined cytokine level or transductionefficiency (or both); and

optionally, selecting the CAR-expressing cell product for administrationto a subject,

thereby manufacturing a CAR-expressing cell product.

In certain embodiments of the aforesaid manufacturing methods, thecytokine is chosen from one, two, three, four, five, six, seven, eight,or more (or all) of CCL20/MIP3a, IL17A, IL6, GM-CSF, IFNγ, IL10, IL13,IL2, IL21, IL4, IL5, IL9 or TNFα, or a combination thereof. In oneembodiment, the cytokine is chosen from one, two, three, four or more(all) of IL-17a, CCL20, IL2, IL6, or TNFa. For example, the cytokine canbe chosen from: one or both of IL-17a and CCL20; one or both ofCCL20/MIP3a and IL17A; or one, two, or all of CCL20/MIP3a, IL17A andIL6. In one embodiment, the cytokine is CCL20/MIP3a. In anotherembodiment, the cytokine is IL17A. In yet another embodiment, thecytokine is IL6.

In certain embodiments of the aforesaid manufacturing methods, atransduction efficiency of 15% or higher is indicative of increasedpotency. In other embodiments, a transduction efficiency of less than15% is indicative of decreased potency.

In certain embodiments, any of the aforesaid manufacturing methodsfurther comprise reducing the number (e.g., depleting) T_(REG) cells,e.g., via CD25-depletion, GITR depletion, or mTOR inhibition.Alternatively, or in combination, the manufacturing methods furthercomprise contacting the sample, e.g., the apheresis sample, with ananti-GITR antibody.

In certain embodiments, any of the aforesaid manufacturing methods eachof (i), (ii) or (iii), (iv), (v), (vi), (vii), (viii), (ix), or anycombination thereof (e.g., all) are evaluated following activation invitro.

In an embodiment, the cytokine profile includes one or more (e.g., one,two, three, four, five, six or more) of CCL20 (also referred to asMIP3a), IL-17a, IL-6, GM-CSF, IFNγ, IL-10, IL-13, IL-2, IL-21, IL-4,IL-5, IL-9 and TNFα. In an embodiment, the cytokine profile includesCCL20. In an embodiment, the cytokine profile includes IL-17a. In anembodiment, the cytokine profile includes IL-6. In one embodiment, thecytokine profile includes two or more (e.g., all three) of CCL20, IL-17aand IL-6.

In one embodiment, the method further includes determining an expressionlevel of one or more cytokines of Table 14, Table 15 or Table 16 (e.g.,CCL20, IL-17a and/or IL-6) secreted by the CAR-expressing cell (e.g., Tcell, NK cell) product. In an embodiment, secretion of one or morecytokines of Table 14, Table 15 or Table 16 (e.g., CCL20, IL-17a and/orIL-6) is in response to CAR-expressing cell product stimulation with oneor more target tumor antigen(s).

In an embodiment, the cytokine signature or level described herein isindicative of the potency of a CAR-expressing cell product. In anembodiment, cytokine signatures described herein are markers of responseto a CAR-expressing cell product in a hematological cancer (e.g., CLLand ALL).

In an embodiment, the cytokine signature or level described hereinpredict subject response to a CAR-expressing cell product.

In an embodiment, the cytokine signature or level described in Table 16predict subject response to a CAR-expressing cell product.

In an embodiment, the IL-17a and CCL-20 expression level predict subjectresponse to a CAR-expressing cell product.

In an embodiment, the method further includes one or more of (e.g., one,two, three or all of): obtaining a blood sample, e.g., a population of Tcells obtained from the blood of a subject; activating the population ofT cells, e.g., by a method described herein; genetically engineering acell from the population of T cells, e.g., transducing a cell from thepopulation of T cells, with a vector comprising a nucleic acid encodinga CAR, e.g., a CAR described herein, e.g., a CD19 CAR described herein,e.g., CTL019; expanding a population of T cells that comprises agenetically engineered T cell, e.g., a cell transduced with a nucleicacid encoding a CAR, e.g., a CAR described herein, e.g., a CD19 CARdescribed herein, e.g., by a method described herein. In an embodiment,the CAR transduction efficiency described herein is indicative ofsubject's response to a CAR-expressing cell therapy in a hematologicalcancer (e.g., CLL and ALL).

In an embodiment, a CAR transduction efficiency described herein ispredictive of subject response to CAR-expressing cell therapy in ahematological disease (e.g., CLL and ALL).

In an embodiment, a cytokine signature or level described herein is usedto improve and/or modify CAR-expressing cell product (e.g., a CD19CAR-expressing cell product such as, e.g., CTL019) prior to infusion inpatients.

In an embodiment, a cytokine signature or level described herein is usedto assess manufactured CAR-expressing cell (e.g., T cell, NK cell)products. In an embodiment, the cytokine signature or level describedherein provide an end point in manufacturing process optimization.

In an embodiment, any of the aforesaid manufacturing methods comprise astep of recording the result of the comparison in a quality controlrecord for the CAR-expressing cell (e.g., T cell, NK cell) preparation(e.g., a CD19 CAR-expressing cell preparation as described herein suchas, e.g., CTL019). In an embodiment, the method further comprisesobtaining a preparation of T cells from a subject identified as apartial responder or non-responder and increasing the number of naïve Tcells in the preparation. In one embodiment, the method furthercomprises introducing a nucleic acid encoding a CAR into the T cellpreparation.

In an embodiment, the method further comprises obtaining a preparationof immune effector cells (e.g., T cells) from a subject identified as apartial responder or non-responder and who has been subsequently treatedwith an agent that increases the number of naïve T cells in the subject,e.g., the subject has been treated with a kinase inhibitor, e.g., anmTOR inhibitor, e.g., as described herein, and/or a checkpointinhibitor, e.g., as described herein. In one embodiment, the methodfurther comprises introducing a nucleic acid encoding a CAR into aplurality of the immune effector cells (e.g., T cells) of thepreparation.

In an embodiment, the CAR-expressing cell (e.g., T cell, NK cell)product is a CD19 CAR-expressing cell, e.g., a CD19 CAR-expressing celldescribed herein, e.g., CTL019.

In still another aspect, the present disclosure provides one or moregene signatures or expression profiles that discriminate relapsers toCAR-expressing cell (e.g., T cell, NK cell) therapy from non-relapsersto CAR-expressing cell therapy in a cancer, e.g., a hematological cancer(e.g., ALL and CLL).

In an embodiment, the one or more gene signatures or expression profilesdescribed herein enable manufactured product improvements, therebyreducing the likelihood of patient relapse. In an embodiment, genesignatures described herein are used to modify therapeutic applicationof manufactured product, thereby reducing the likelihood of patientrelapse.

In an embodiment, the one or more gene signatures or expression profilesdescribed herein are identified in a subject prior to CAR-expressingcell treatment (e.g., a CD19 CAR-expressing cell treatment, e.g., CTL019therapy) that predict relapse to CAR-expressing cell treatment. In anembodiment, the one or more gene signatures or expression profilesdescribed herein are identified in an apheresis sample. In anembodiment, the one or more gene signatures or expression profilesdescribed herein are identified in a bone marrow sample. In anembodiment, the one or more gene signatures or expression profilesdescribed herein are identified in a manufactured CAR-expressing cellproduct (e.g., a CD19 CAR-expressing cell product, e.g., CTL019) priorto infusion.

In an embodiment, decreasing the T_(REG) level or gene signature in asubject prior to apheresis or during manufacturing of a CAR-expressingcell product significantly reduces the risk of subject relapse.

In an embodiment, a subject is pre-treated with one or more therapiesthat reduce T_(REG) cells prior to collection of cells forCAR-expressing cell product manufacturing, thereby reducing the risk ofsubject relapse to CAR-expressing cell treatment (e.g., CTL019treatment). In an embodiment, methods of decreasing T_(REG) cellsinclude, but are not limited to, administration to the subject of one ormore of cyclophosphamide, anti-GITR antibody, CD25-depletion, mTORinhibitor, or a combination thereof. Administration of one or more ofcyclophosphamide, anti-GITR antibody, CD25-depletion, mTOR inhibitor, ora combination thereof, can occur before, during or after an infusion ofthe CAR-expressing cell product.

In an embodiment, a subject is pre-treated with cyclophosphamide priorto collection of cells for CAR-expressing cell product manufacturing,thereby reducing the risk of subject relapse to CAR-expressing celltreatment (e.g., CTL019 treatment).

In an embodiment, a subject is pre-treated with an anti-GITR antibodyprior to collection of cells for CAR-expressing cell productmanufacturing, thereby reducing the risk of subject relapse toCAR-expressing cell treatment (e.g., CTL019 treatment).

In an embodiment, a CAR-expressing cell manufacturing process ismodified to deplete T_(REG) cells prior to manufacturing of theCAR-expressing cell product (e.g., a CTL019 product). In an embodiment,CD25-depletion is used to deplete T_(REG) cells prior to manufacturingof the CAR-expressing cell product (e.g., a CTL019 product).Accordingly, in some embodiments, the method further comprises:

a. providing a population of immune effector cells (e.g., T cells or NKcells); and

b. removing T regulatory cells from the population, thereby providing apopulation of T regulatory-depleted cells;

wherein steps a) and b) are performed prior to introducing the nucleicacid encoding the CAR to the population.

In embodiments of the methods, the T regulatory cells comprise CD25+ Tcells, and are removed from the cell population using an anti-CD25antibody, or fragment thereof. The anti-CD25 antibody, or fragmentthereof, can be conjugated to a substrate, e.g., a bead.

In other embodiments, the population of T regulatory-depleted cellsprovided from step (b) contains less than 30%, 25%, 20%, 15%, 10%, 5%,4%, 3%, 2%, 1% of CD25+ cells.

In yet other embodiments, the method further comprises removing cellsfrom the population which express a tumor antigen that does not compriseCD25 to provide a population of T regulatory-depleted and tumor antigendepleted cells prior to introducing the nucleic acid encoding a CAR tothe population. The tumor antigen can be selected from CD19, CD30, CD38,CD123, CD20, CD14 or CD11b, or a combination thereof.

In other embodiments, the method further comprises removing cells fromthe population which express a checkpoint inhibitor, to provide apopulation of T regulatory-depleted and inhibitory molecule depletedcells prior to introducing the nucleic acid encoding a CAR to thepopulation. An inhibitory molecule, e.g., a checkpoint inhibitor, can bechosen from PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1,CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4,CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR,A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR (e.g.,TGFRbeta), e.g., as described herein.

Further embodiments disclosed herein encompass providing a population ofimmune effector cells. The population of immune effector cells providedcan be selected based upon the expression of one or more of CD3, CD28,CD4, CD8, CD45RA, and/or CD45RO. In certain embodiments, the populationof immune effector cells provided are CD3+ and/or CD28+.

In certain embodiments of the method, the method further comprisesexpanding the population of cells after the nucleic acid moleculeencoding a CAR has been introduced.

In embodiments, the population of cells is expanded for a period of 8days or less.

In certain embodiments, the population of cells is expanded in culturefor 5 days, and the resulting cells are more potent than the same cellsexpanded in culture for 9 days under the same culture conditions.

In other embodiments, the population of cells is expanded in culture for5 days, and shows at least a one, two, three or four fold increase incell doublings upon antigen stimulation as compared to the same cellsexpanded in culture for 9 days under the same culture conditions.

In yet other embodiments, the population of cells is expanded in culturefor 5 days, and the resulting cells exhibit higher proinflammatory IFN-γand/or GM-CSF levels, as compared to the same cells expanded in culturefor 9 days under the same culture conditions.

In other embodiments, the population of cells is expanded by culturingthe cells in the presence of an agent that stimulates a CD3/TCR complexassociated signal and/or a ligand that stimulates a costimulatorymolecule on the surface of the cells. The agent can be a bead conjugatedwith anti-CD3 antibody, or a fragment thereof, and/or anti-CD28antibody, or a fragment thereof.

In other embodiments, the population of cells is expanded in anappropriate media that includes one or more interleukin that result inat least a 200-fold, 250-fold, 300-fold, or 350-fold increase in cellsover a 14 day expansion period, as measured by flow cytometry.

In other embodiments, the population of cells is expanded in thepresence IL-15 and/or IL-7.

In certain embodiments, the method further includes cryopreserving thepopulation of the cells after the appropriate expansion period.

In yet other embodiments, the method of making disclosed herein furthercomprises contacting the population of immune effector cells with anucleic acid encoding a telomerase subunit, e.g., hTERT. The nucleicacid encoding the telomerase subunit can be DNA.

In yet other embodiments, the method of making disclosed herein furthercomprises culturing the population of immune effector cells in serumcomprising 2% hAB serum.

In any of the methods, systems and kits described herein, the CD19 CARcan comprise an anti-CD19 binding domain described in Table 12, or CDRs,e.g., one or more (e.g., all) of HC CDR1, HC CDR2, HC CDR3, LC CDR1, LCCDR2, and LC CDR3 of an anti-CD19 binding domain described in Table 12.In an embodiment, the CAR can comprise one of more of: a leadersequence, e.g., a leader sequence described herein, e.g., in Table 11;an anti-CD19 binding domain, e.g., an anti-CD19 binding domain describedherein, e.g., in Table 12; a hinge region, e.g., a hinge regiondescribed herein, e.g., a hinge region described in Table 11; atransmembrane domain, e.g., a transmembrane domain described herein,e.g., in Table 11; and an intracellular signaling domain (e.g., acostimulatory domain and/or a primary signaling domain, e.g., acostimulatory domain described herein, e.g., in Table 11 and/or aprimary signaling domain described herein, e.g., in Table 11). In anembodiment, the CD19 CAR-expressing cell (e.g., T cell, NK cell) isCTL019 or a CD19 CAR described in Table 13.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references (e.g., sequencedatabase reference numbers) mentioned herein are incorporated byreference in their entirety. For example, all GenBank, Unigene, andEntrez sequences referred to herein, e.g., in any Table herein, e.g., inany of Table 1A, Table 1B, Table 7A, Table 7B, Table 8, Table 14, Table17, Table 18, and Table 20, are incorporated by reference. Unlessotherwise specified, the sequence accession numbers specified herein,including in any Table herein, e.g., in any of Table 1A, Table 1B, Table7A, Table 7B, Table 8, Table 14, Table 17, Table 18, and Table 20, referto the database entries current as of Oct. 8, 2014. When one gene orprotein references a plurality of sequence accession numbers, all of thesequence variants are encompassed.

In addition, the materials, methods, and examples are illustrative onlyand not intended to be limiting. Headings, sub-headings or numbered orlettered elements, e.g., (a), (b), (i) etc., are presented merely forease of reading. The use of headings or numbered or lettered elements inthis document does not require the steps or elements be performed inalphabetical order or that the steps or elements are necessarilydiscrete from one another. Other features, objects, and advantages ofthe invention will be apparent from the description and drawings, andfrom the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an exemplary model illustrating findings from a wholegenome CTL019 RNAseq analysis performed on T cells at apheresis andafter CTL019 manufacturing for 21 CLL and 7 ALL samples. This modeldemonstrates that expression patterns of complete responders (CRs) havea younger T cell phenotype than non-responders (NRs). Memory T cellsubsets are differentially enriched between CRs versus NRs with CRsshowing similarity to T memory stem cells.

FIG. 2A depicts an exemplary histogram of the number of samples analyzedin the whole genome CTL019 RNAseq analysis described herein. p=product;a=apheresis. FIG. 2B is an exemplary schematic illustrating an overviewof the analysis. Briefly, for each gene set, a 3-group statistical modelwas applied to determine whether the meta-gene was statisticallydifferent between the CLL product CRs, PRs, and NRs. CRs are more likeresting T_(EFF) cells, whereas NR are more like activated T_(EFF) cells.CTL019 NR samples are in a more activated state than CR samples.

FIG. 3 depicts an exemplary schematic of memory T cell precursors andsubsets. Without wishing to be bound by a particular theory, the stateof memory T cells in CTL019 samples is likely a major component ofresponse.

FIG. 4 depicts an exemplary result illustrating meta-gene scores for theT_(SCM) vs T_(CM) upregulated gene set. The x-axis is samples byresponse group where a=apheresis and p=product. The y-axis is normalizedmeta-gene expression scores. Gene sets enriched in CLL CRs (e.g., CTL019CRs) are also enriched in acute lymphoblastic leukemias (ALLs). ALL andCLL CRs are enriched in T stem cell (T_(SCM)) subset specific genes,whereas CLL PRs and NRs are enriched in T central memory (T_(CM)) subsetgenes. The same pattern is seen in apheresis as in product samples. ALLexpression patters are most similar to CLL CRs and are even more extremein the direction of resting/unstimulated/early memory T cells.

FIG. 5A depicts an exemplary result from a Principle Component Analysis(PCA) of CTL019 samples. This exemplary PCA result illustrates that CRs,ALL and Normal samples cluster separately from PRs and NRs. FIG. 5Bdepicts an exemplary result from a PCA of CTL019 and apheresis samples.This exemplary PCA result illustrates that CRs, ALL and Normal samplescluster separately from PRs and NRs and from the apheresis cluster.

FIG. 6 depicts an exemplary schematic depicting immunophenotyping ofapheresis and product samples.

FIG. 7A and FIG. 7B depict exemplary multi-color flow cytometry analysisresults identifying correlates of response in product samples. 36manufactured CTL019 samples from CLL patients were analyzed. Samplesincluded 5 CR, 8 PR, 19NR and 3 pending. FIG. 7A depicts an exemplaryresult illustrating percent CD4+ cells and patient response. FIG. 7Bdepicts an exemplary result illustrating percent CD8+ cells and patientresponse.

FIG. 8A, FIG. 8B, FIG. 8C and FIG. 8D depicts an exemplary flowcytometry analysis of PD1 and CAR19 expression on T cells. FIG. 8A andFIG. 8B are representative flow cytometry profiles demonstrating thedistribution of PD-1 and CAR19 expression on CD4+ T cells from subjectsthat are complete responders (CR) or non-responders (NR) toCAR-expressing cell therapy. FIG. 8C is a graph showing the percent ofPD1 cells in the CD4+ T cell population from groups of subjects withdifferent responses to CAR-expressing cell therapy. FIG. 8D is a graphshowing the percent of PD1 cells in the CD8+ T cell population fromgroups of subjects with different responses to CAR-expressing celltherapy.

FIG. 9A and FIG. 9B depict an exemplary flow cytometry analysis of PD1,CAR 19, and LAG-3 expression on T cells from subjects that are completeresponders (CR) or non-responders (NR) to CAR-expressing cell therapy.FIG. 9C depicts exemplary results that show the distribution of PD1 andLAG-3 expression from groups of subjects with different responses toCAR-expressing cell therapy. Non-responder (NR) products have higherpercentages of PD1+CAR19+LAG3+ T cells than CRs. These data demonstratethat NR products exhibit an exhausted phenotype of PD1+CAR+ andco-expression of LAG3.

FIG. 10A and FIG. 10B depict an exemplary flow cytometry analysis ofPD1, CAR 19, and TIM-3 expression on T cells from subjects that arecomplete responders (CR) or non-responders (NR) to CAR-expressing celltherapy. FIG. 10C depicts exemplary results that show the distributionof PD1 and TIM-3 expression from groups of subjects with differentresponses to CAR-expressing cell therapy. Non-responder (NR) productshave higher percentages of CAR19+PD1+TIM3+ cells than CRs. These datademonstrate that NR products exhibit an exhausted phenotype of PD1+CAR+and co-expression of TIM3.

FIG. 11 depicts an exemplary result illustrating that CD27 levels in theCAR product correlate with patient response. CRs CD8+ cells displayed ahigher percentage of CD27+ cells as compared to PRs and NRs.

FIG. 12 depicts an exemplary multi-color flow cytometry analysis resultidentifying correlates of response in apheresis samples. 26 apheresedsamples from CLL patients were analyzed. Samples included 4 CR, 6 PR,14NR and 1 patient was not infused.

FIG. 13 depicts an exemplary multi-color flow cytometry analysis resultillustrating a correlation between a younger T cell phenotype andresponse to CTL019 therapy. These data demonstrate that the percentageof CD27+CD45RO− in CD8+ T cells is predictive of which CLL patients willundergo a complete response to CTL019.

FIG. 14 depicts an exemplary analysis of apheresis in a human patientprior to CTL019 therapy. Exemplary results illustrate that while patient1000-00045 presented with very few T cells, 27% of the T cells wereCD8+CD27+CD45RO−.

FIG. 15 depicts an exemplary result of a patient response (patient1000-00045) to CTL019 therapy. CD8+CD27+CD45RO− T cells were a positivepredictor of the patient response to CTL019 therapy. These exemplaryresults illustrate that a good prognostic phenotype in apheresis is ahigh percentage of CD8+CD27+CD45RO− T cells (naïve or T_(SCM)phenotype). A poor prognostic phenotype in CTL019 product is a highpercentage of PD1+CAR+ and LAG3+ or TIM3+ T cells (exhausted phenotype).

FIG. 16 depicts an exemplary block diagram of a computer system on whichvarious aspects and embodiments may be practiced.

FIG. 17 depicts an exemplary heatmap showing bi-clustering of cytokineexpression in stimulated CTL019 products and CLL patients. Two clusters(Cluster 1 and Cluster 3) were almost exclusively comprised of CRs andPRs, whereas the other two clusters (Cluster 2 and Cluster 4) containedpredominantly NRs. On average, cytokine expression levels were higher inCRs/PRs versus NRs. The top panel shows the red channel of the heatmapimage, the center panel shows the blue channel, and the bottom panelshows the green channel.

FIG. 18 depicts exemplary results of log-normalized expressions ofstatistically significant cytokines (e.g., CCL20/MIP3a, IL2, TNFα, IL17aand IL6) to distinguish CRs, PRs and NRs in CLL patients.

FIG. 19A depicts an exemplary scatter plot showing log-normalizedcorrelation of IL17A (y-axis) and CCL20 (x-axis) expression. Dashedlines represent the classification boundary for separating NRs fromCRs/PRs. Each dot represents a CLL patient, and the cross-hatch (NR),black (PR) and white (CR) represent the clinical response. Thecorrelation coefficient is represented by “r” (e.g., a correlationcoefficient of 0.928) and corresponding p-value for correlation using“p.value” (e.g., corresponding p-value of 1.36e-09). FIG. 19B depicts anexemplary scatter plot showing correlation of CCL20 with percentage ofCAR+ cells with a correlation coefficient of 0.395 and correspondingp-value of 0.0761. Each dot represents a CLL patient, and thecross-hatch (NR), black (PR) and white (CR) represent the clinicalresponse. The correlation coefficient is represented by “r” andcorresponding p-value for correlation using “p.value”. FIG. 19C depictsan exemplary scatter plot showing correlation of IL17a with percentageof CAR+ cells with a correlation coefficient of 0.278 and correspondingp-value of 0.222. Each dot represents a CLL patient, and the cross-hatch(NR), black (PR) and white (CR) represent the clinical response. Thecorrelation coefficient is represented by “r” and corresponding p-valuefor correlation using “p.value”.

FIG. 20 depicts exemplary results (p=0.000215) illustrating that T_(REG)genes have high expression levels in relapsers (R) compared tonon-relapser, complete responders (CR). The x-axis is samples byresponse group where CR=complete responder and R=relapser. The y-axis isnormalized meta-gene expression scores.

FIG. 21 depicts an exemplary scatter plot showing percent of CAR+ cells(i.e., transduction rate) at pre-harvest for complete responders (CR) inwhite, partial responders (PR) in black and non-responders (NR) inhatching. Transduction efficiencies were measured pre-harvest andcorrelated with subject response (e.g., CR, PR, or NR). The solid linerepresents a 15% transduction efficiency that separates the majority ofnon-responders from responders. Without wishing to be bound to aparticular theory, these data indicate that pre-harvest CAR transductionrate is a marker of response to CAR-expressing cell (e.g., T cell, NKcell) therapy in CLL.

FIG. 22 is a bar graph depicting the relationship between number ofCD27+PD1− CART cells infused and response to therapy.

FIG. 23 is a scatter plot depicting the relationship between number ofCD27+PD1-CART cells infused and response to therapy.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains.

The term “a” and “an” refers to one or to more than one (i.e., to atleast one) of the grammatical object of the article. By way of example,“an element” means one element or more than one element.

The term “about” when referring to a measurable value such as an amount,a temporal duration, and the like, is meant to encompass variations of±20% or in some instances ±10%, or in some instances ±5%, or in someinstances ±1%, or in some instances ±0.1% from the specified value, assuch variations are appropriate to perform the disclosed methods.

“Acquire” or “acquiring” as the terms are used herein, refer toobtaining possession of a physical entity (e.g., a sample, apolypeptide, a nucleic acid, or a sequence), or a value, e.g., anumerical value, by “directly acquiring” or “indirectly acquiring” thephysical entity or value. “Directly acquiring” means performing aprocess (e.g., performing a synthetic or analytical method) to obtainthe physical entity or value. “Indirectly acquiring” refers to receivingthe physical entity or value from another party or source (e.g., a thirdparty laboratory that directly acquired the physical entity or value).Directly acquiring a physical entity includes performing a process thatincludes a physical change in a physical substance, e.g., a startingmaterial. Exemplary changes include making a physical entity from two ormore starting materials, shearing or fragmenting a substance, separatingor purifying a substance, combining two or more separate entities into amixture, performing a chemical reaction that includes breaking orforming a covalent or non-covalent bond. Directly acquiring a valueincludes performing a process that includes a physical change in asample or another substance, e.g., performing an analytical processwhich includes a physical change in a substance, e.g., a sample,analyte, or reagent (sometimes referred to herein as “physicalanalysis”), performing an analytical method, e.g., a method whichincludes one or more of the following: separating or purifying asubstance, e.g., an analyte, or a fragment or other derivative thereof,from another substance; combining an analyte, or fragment or otherderivative thereof, with another substance, e.g., a buffer, solvent, orreactant; or changing the structure of an analyte, or a fragment orother derivative thereof, e.g., by breaking or forming a covalent ornon-covalent bond, between a first and a second atom of the analyte; orby changing the structure of a reagent, or a fragment or otherderivative thereof, e.g., by breaking or forming a covalent ornon-covalent bond, between a first and a second atom of the reagent.

The term “antibody,” as used herein, refers to a protein, or polypeptidesequence derived from an immunoglobulin molecule which specificallybinds with an antigen. Antibodies can be polyclonal or monoclonal,multiple or single chain, or intact immunoglobulins, and may be derivedfrom natural sources or from recombinant sources. Antibodies can betetramers of immunoglobulin molecules.

The term “altered level of expression” of a biomarker as describedherein (e.g., a biomarker listed in Table 1A, Table 1B, Table 7A, Table7B, Table 8, Table 9, Table 10, Table 14, Table 15, Table 16, Table 18,Table 20, PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L, KLRG1, and aCD19 CAR-expressing cell (e.g., T cell, NK cell) gene signature) refersto an increase (or decrease) in the expression level of a marker in atest sample, such as a sample derived from a patient suffering fromcancer (e.g., a hematological cancer such as ALL and CLL) that isgreater or less than the standard error of the assay employed to assessexpression. In embodiments, the alteration can be at least twice, atleast twice three, at least twice four, at least twice five, or at leasttwice ten or more times greater than or less than the expression levelof the biomarkers in a control sample (e.g., a sample from a healthysubject not having the associated disease), or the average expressionlevel in several control samples. An “altered level of expression” canbe determined at the protein or nucleic acid (e.g., mRNA) level.

The term “antibody fragment” refers to at least one portion of anantibody, that retains the ability to specifically interact with (e.g.,by binding, steric hindrance, stabilizing/destabilizing, spatialdistribution) an epitope of an antigen. Examples of antibody fragmentsinclude, but are not limited to, Fab, Fab, F(ab)₂, Fv fragments, scFvantibody fragments, disulfide-linked Fvs (sdFv), a Fd fragmentconsisting of the VH and CH1 domains, linear antibodies, single domainantibodies such as sdAb (either VL or VH), camelid VHH domains,multi-specific antibodies formed from antibody fragments such as abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region, and an isolated CDR or other epitope bindingfragments of an antibody. An antigen binding fragment can also beincorporated into single domain antibodies, maxibodies, minibodies,nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR andbis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology23:1126-1136, 2005). Antigen binding fragments can also be grafted intoscaffolds based on polypeptides such as a fibronectin type III (Fn3)(seeU.S. Pat. No. 6,703,199, which describes fibronectin polypeptideminibodies). The term “scFv” refers to a fusion protein comprising atleast one antibody fragment comprising a variable region of a lightchain and at least one antibody fragment comprising a variable region ofa heavy chain, wherein the light and heavy chain variable regions arecontiguously linked via a short flexible polypeptide linker, and capableof being expressed as a single chain polypeptide, and wherein the scFvretains the specificity of the intact antibody from which it is derived.Unless specified, as used herein an scFv may have the VL and VH variableregions in either order, e.g., with respect to the N-terminal andC-terminal ends of the polypeptide, the scFv may comprise VL-linker-VHor may comprise VH-linker-VL.

The term “antibody heavy chain,” refers to the larger of the two typesof polypeptide chains present in antibody molecules in their naturallyoccurring conformations, and which normally determines the class towhich the antibody belongs.

The term “antibody light chain,” refers to the smaller of the two typesof polypeptide chains present in antibody molecules in their naturallyoccurring conformations. Kappa (κ) and lambda (λ) light chains refer tothe two major antibody light chain isotypes.

The term “complementarity determining region” or “CDR,” as used herein,refers to the sequences of amino acids within antibody variable regionswhich confer antigen specificity and binding affinity. For example, ingeneral, there are three CDRs in each heavy chain variable region (e.g.,HCDR1, HCDR2, and HCDR3) and three CDRs in each light chain variableregion (LCDR1, LCDR2, and LCDR3). The precise amino acid sequenceboundaries of a given CDR can be determined using any of a number ofwell-known schemes, including those described by Kabat et al. (1991),“Sequences of Proteins of Immunological Interest,” 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (“Kabat” numberingscheme), Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numberingscheme), or a combination thereof Under the Kabat numbering scheme, insome embodiments, the CDR amino acid residues in the heavy chainvariable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and95-102 (HCDR3); and the CDR amino acid residues in the light chainvariable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and89-97 (LCDR3). Under the Chothia numbering scheme, in some embodiments,the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2),and 95-102 (HCDR3); and the CDR amino acid residues in the VL arenumbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In a combinedKabat and Chothia numbering scheme, in some embodiments, the CDRscorrespond to the amino acid residues that are part of a Kabat CDR, aChothia CDR, or both. For instance, in some embodiments, the CDRscorrespond to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and95-102 (HCDR3) in a VH, e.g., a mammalian VH, e.g., a human VH; andamino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in aVL, e.g., a mammalian VL, e.g., a human VL.

The term “anti-cancer effect” refers to a biological effect which can bemanifested by various means, including but not limited to, e.g., adecrease in tumor volume, a decrease in the number of cancer cells, adecrease in the number of metastases, an increase in life expectancy,decrease in cancer cell proliferation, decrease in cancer cell survival,or amelioration of various physiological symptoms associated with thecancerous condition. An “anti-cancer effect” can also be manifested bythe ability of the peptides, polynucleotides, cells and antibodies inprevention of the occurrence of cancer in the first place. The term“anti-tumor effect” refers to a biological effect which can bemanifested by various means, including but not limited to, e.g., adecrease in tumor volume, a decrease in the number of tumor cells, adecrease in tumor cell proliferation, or a decrease in tumor cellsurvival.

The term “allogeneic” refers to any material derived from a differentanimal of the same species as the individual to whom the material isintroduced. Two or more individuals are said to be allogeneic to oneanother when the genes at one or more loci are not identical. In someaspects, allogeneic material from individuals of the same species may besufficiently unlike genetically to interact antigenically.

The term “apheresis” as used herein refers to an extracorporeal processby which the blood of a donor or patient is removed from the donor orpatient and passed through an apparatus that separates out selectedparticular constituent(s) and returns the remainder to the circulationof the donor or patient, e.g., by retransfusion. Thus, in the context of“an apheresis sample” refers to a sample obtained using apheresis.

The term “autologous” refers to any material derived from the sameindividual to whom it is later to be re-introduced into the individual.

A “biomarker” or “marker” is a gene, mRNA, or protein that undergoesalterations in expression that are associated with progression of cancer(e.g., a hematological cancer such as ALL and CLL) or responsiveness totreatment. The alteration can be in amount and/or activity in abiological sample (e.g., a blood, plasma, or a serum sample) obtainedfrom a subject having cancer, as compared to its amount and/or activity,in a sample obtained from a baseline or prior value for the subject, thesubject at a different time interval, an average or median value for acancer patient population, a healthy control, or a healthy subjectpopulation (e.g., a control); such alterations in expression and/oractivity are associated with of the responsiveness of a subject having acancer disease state (e.g., a hematological cancer such as ALL and CLL)to a CAR-expressing cell (e.g., a CAR-expressing immune effector cell(e.g., a CAR-expressing T cell, NK cell) therapy, e.g., a CD19CAR-expressing cell therapy. For example, a marker of the inventionwhich is predictive of responsiveness to therapeutics can have analtered expression level, protein level, or protein activity, in abiological sample obtained from a subject having, or suspected ofhaving, cancer as compared to a biological sample obtained from acontrol subject.

The term “cancer” refers to a disease characterized by the uncontrolledgrowth of aberrant cells. Cancer cells can spread locally or through thebloodstream and lymphatic system to other parts of the body. Examples ofvarious cancers are described herein and include but are not limited to,breast cancer, prostate cancer, ovarian cancer, cervical cancer, skincancer, pancreatic cancer, colorectal cancer, renal cancer, livercancer, brain cancer, lymphoma, leukemia, lung cancer and the like.Cancers include, but are not limited to, B-cell acute lymphocyticleukemia (B-ALL), T-cell acute lymphocytic leukemia (T-ALL), acutelymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), chroniclymphocytic leukemia (CLL), B cell promyelocytic leukemia, blasticplasmacytoid dendritic cell neoplasm, Burkitts lymphoma, diffuse large Bcell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- ora large cell-follicular lymphoma, malignant lymphoproliferativeconditions, MALT lymphoma, mantle cell lymphoma (MCL), marginal zonelymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome,non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma,plasmacytoid dendritic cell neoplasm, and Waldenstrom macroglobulinemia.In an embodiment, the cancer is associated with CD19 expression. Theterms “tumor” and “cancer” are used interchangeably herein, e.g., bothterms encompass solid and liquid tumors. As used herein, the term“cancer” or “tumor” includes premalignant, as well as malignant cancersand tumors.

The terms “cancer associated antigen” or “tumor antigen” interchangeablyrefers to a molecule (typically protein, carbohydrate or lipid) that ispreferentially expressed on the surface of a cancer cell, eitherentirely or as a fragment (e.g., MHC/peptide), in comparison to a normalcell, and which is useful for the preferential targeting of apharmacological agent to the cancer cell. In some embodiments, a tumorantigen is a marker expressed by both normal cells and cancer cells,e.g., a lineage marker, e.g., CD19 on B cells. In some embodiments, acancer-associated antigen is a cell surface molecule that isoverexpressed in a cancer cell in comparison to a normal cell, forinstance, 1-fold over expression, 2-fold overexpression, 3-foldoverexpression or more in comparison to a normal cell. In someembodiments, a cancer-associated antigen is a cell surface molecule thatis inappropriately synthesized in the cancer cell, for instance, amolecule that contains deletions, additions or mutations in comparisonto the molecule expressed on a normal cell. In some embodiments, a tumorantigen will be expressed exclusively on the cell surface of a cancercell, entirely or as a fragment (e.g., MHC/peptide), and not synthesizedor expressed on the surface of a normal cell. In some embodiments, theCARs of the present invention includes CARs comprising an antigenbinding domain (e.g., antibody or antibody fragment) that binds to a MHCpresented peptide. Normally, peptides derived from endogenous proteinsfill the pockets of Major histocompatibility complex (MHC) class Imolecules, and are recognized by T cell receptors (TCRs) on CD8+Tlymphocytes. The MHC class I complexes are constitutively expressed byall nucleated cells. In cancer, virus-specific and/or tumor-specificpeptide/MHC complexes represent a unique class of cell surface targetsfor immunotherapy. TCR-like antibodies targeting peptides derived fromviral or tumor antigens in the context of human leukocyte antigen(HLA)-A1 or HLA-A2 have been described (see, e.g., Sastry et al., JVirol. 2011 85(5):1935-1942; Sergeeva et al., Blood, 2011117(16):4262-4272; Verma et al., J Immunol 2010 184(4):2156-2165;Willemsen et al., Gene Ther 2001 8(21):1601-1608; Dao et al., Sci TranslMed 2013 5(176):176ra33; Tassev et al., Cancer Gene Ther 201219(2):84-100). For example, TCR-like antibody can be identified fromscreening a library, such as a human scFv phage displayed library.

As used herein, the term “CD19” refers to the Cluster of Differentiation19 protein, which is an antigenic determinant detectable on leukemiaprecursor cells. The human and murine amino acid and nucleic acidsequences can be found in a public database, such as GenBank, UniProtand Swiss-Prot. For example, the amino acid sequence of human CD19 canbe found as UniProt/Swiss-Prot Accession No. P15391 and the nucleotidesequence encoding of the human CD19 can be found at Accession No.NM_001178098. As used herein, “CD19” includes proteins comprisingmutations, e.g., point mutations, fragments, insertions, deletions andsplice variants of full length wild-type CD19. CD19 is expressed on mostB lineage cancers, including, e.g., acute lymphoblastic leukemia,chronic lymphocyte leukemia and non-Hodgkin lymphoma. Other cells whichexpress CD19 are provided below in the definition of “disease associatedwith expression of CD19.” It is also an early marker of B cellprogenitors. See, e.g., Nicholson et al., MOL. IMMUN. 34 (16-17):1157-1165 (1997). In one aspect the antigen-binding portion of theCAR-expressing cell (e.g., T cell, NK cell) recognizes and binds anantigen within the extracellular domain of the CD19 protein. In oneaspect, the CD19 protein is expressed on a cancer cell. In oneembodiment, the CD19 has a wild-type sequence, e.g., a wild-type humansequence. In another embodiment, the CD19 has a mutant sequence, e.g., amutant human sequence.

The term “Chimeric Antigen Receptor” or alternatively a “CAR” refers toa set of polypeptides, typically two in the simplest embodiments, whichwhen in an immune effector cell, provides the cell with specificity fora target cell, typically a cancer cell, and with intracellular signalgeneration. In some embodiments, a CAR comprises at least anextracellular antigen binding domain, a transmembrane domain and acytoplasmic signaling domain (also referred to herein as “anintracellular signaling domain”) comprising a functional signalingdomain derived from a stimulatory molecule and/or costimulatory moleculeas defined below. In some embodiments, the set of polypeptides are inthe same polypeptide chain (e.g., comprise a chimeric fusion protein).In some embodiments, the set of polypeptides are not contiguous witheach other, e.g., are in different polypeptide chains. In some aspects,the set of polypeptides include a dimerization switch that, upon thepresence of a dimerization molecule, can couple the polypeptides to oneanother, e.g., can couple an antigen binding domain to an intracellularsignaling domain. In one aspect, the stimulatory molecule of the CAR isthe zeta chain associated with the T cell receptor complex. In oneaspect, the cytoplasmic signaling domain comprises a primary signalingdomain (e.g., a primary signaling domain of CD3-zeta). In one aspect,the cytoplasmic signaling domain further comprises one or morefunctional signaling domains derived from at least one costimulatorymolecule as defined below. In one aspect, the costimulatory molecule ischosen from 4-1BB (i.e., CD137), CD27, ICOS, and/or CD28. In one aspect,the CAR comprises a chimeric fusion protein comprising an extracellularantigen recognition domain, a transmembrane domain and an intracellularsignaling domain comprising a functional signaling domain derived from astimulatory molecule. In one aspect, the CAR comprises a chimeric fusionprotein comprising an extracellular antigen recognition domain, atransmembrane domain and an intracellular signaling domain comprising afunctional signaling domain derived from a co-stimulatory molecule and afunctional signaling domain derived from a stimulatory molecule. In oneaspect, the CAR comprises a chimeric fusion protein comprising anextracellular antigen recognition domain, a transmembrane domain and anintracellular signaling domain comprising two functional signalingdomains derived from one or more co-stimulatory molecule(s) and afunctional signaling domain derived from a stimulatory molecule. In oneaspect, the CAR comprises a chimeric fusion protein comprising anextracellular antigen recognition domain, a transmembrane domain and anintracellular signaling domain comprising at least two functionalsignaling domains derived from one or more co-stimulatory molecule(s)and a functional signaling domain derived from a stimulatory molecule.In one aspect the CAR comprises an optional leader sequence at theamino-terminus (N-ter) of the CAR fusion protein. In one aspect, the CARfurther comprises a leader sequence at the N-terminus of theextracellular antigen recognition domain, wherein the leader sequence isoptionally cleaved from the antigen recognition domain (e.g., a scFv)during cellular processing and localization of the CAR to the cellularmembrane. In an embodiment, the CAR is CTL019.

The portion of the CAR composition comprising an antibody or antibodyfragment thereof may exist in a variety of forms where the antigenbinding domain is expressed as part of a contiguous polypeptide chainincluding, for example, a single domain antibody fragment (sdAb), asingle chain antibody (scFv) and a humanized antibody (Harlow et al.,1999, In: USING ANTIBODIES: A LABORATORY MANUAL, COLD SPRING HARBORLABORATORY PRESS, NY; Harlow et al., 1989, In: ANTIBODIES: A LABORATORYMANUAL, Cold Spring Harbor, N.Y.; Houston et al., 1988, PROC. NATL.ACAD. SCI. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). Inone aspect, the antigen binding domain of a CAR composition of theinvention comprises an antibody fragment. In a further aspect, the CARcomprises an antibody fragment that comprises a scFv.

As used herein, the term “binding domain” or “antibody molecule” refersto a protein, e.g., an immunoglobulin chain or fragment thereof,comprising at least one immunoglobulin variable domain sequence. Theterm “binding domain” or “antibody molecule” encompasses antibodies andantibody fragments. In an embodiment, an antibody molecule is amultispecific antibody molecule, e.g., it comprises a plurality ofimmunoglobulin variable domain sequences, wherein a first immunoglobulinvariable domain sequence of the plurality has binding specificity for afirst epitope and a second immunoglobulin variable domain sequence ofthe plurality has binding specificity for a second epitope. In anembodiment, a multispecific antibody molecule is a bispecific antibodymolecule. A bispecific antibody has specificity for no more than twoantigens. A bispecific antibody molecule is characterized by a firstimmunoglobulin variable domain sequence which has binding specificityfor a first epitope and a second immunoglobulin variable domain sequencethat has binding specificity for a second epitope.

The portion of the CAR of the invention comprising an antibody orantibody fragment thereof may exist in a variety of forms where theantigen binding domain is expressed as part of a contiguous polypeptidechain including, for example, a single domain antibody fragment (sdAb),a single chain antibody (scFv), a humanized antibody, or bispecificantibody (Harlow et al., 1999, In: Using Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989,In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houstonet al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al.,1988, Science 242:423-426). In one aspect, the antigen binding domain ofa CAR composition of the invention comprises an antibody fragment. In afurther aspect, the CAR comprises an antibody fragment that comprises ascFv.

The phrase “disease associated with expression of CD19” includes, but isnot limited to, a disease associated with expression of CD19 (e.g., wildtype or mutant CD19) or condition associated with cells which express,or at any time expressed, CD19 including, e.g., proliferative diseasessuch as a cancer or malignancy or a precancerous condition such as amyelodysplasia, a myelodysplastic syndrome or a preleukemia; or anoncancer related indication associated with cells which express CD19.For the avoidance of doubt, a disease associated with expression of CD19may include a condition associated with cells which do not presentlyexpress CD19, e.g., because CD19 expression has been downregulated,e.g., due to treatment with a molecule targeting CD19, e.g., a CD19 CAR,but which at one time expressed CD19. In one aspect, a cancer associatedwith expression of CD19 is a hematological cancer. In one aspect, thehematological cancer is a leukemia or a lymphoma. In one aspect, acancer associated with expression of CD19 includes cancers andmalignancies including, but not limited to, e.g., one or more acuteleukemias including but not limited to, e.g., acute myeloid leukemia(AML), B-cell acute lymphocytic leukemia (“B-ALL”), T-cell acutelymphocytic leukemia (“T-ALL”), acute lymphocytic leukemia (ALL); one ormore chronic leukemias including but not limited to, e.g., chronicmyelogenous leukemia (CML), chronic lymphocytic leukemia (CLL).Additional cancers or hematologic conditions associated with expressionof CD19 comprise, but are not limited to, e.g., B cell promyelocyticleukemia, blastic plasmacytoid dendritic cell neoplasm, Burkittslymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cellleukemia, small cell- or a large cell-follicular lymphoma, malignantlymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma(MCL), marginal zone lymphoma, multiple myeloma, myelodysplasia andmyelodysplastic syndrome, non-Hodgkin lymphoma, Hodgkin lymphoma,plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm,Waldenstrom macroglobulinemia, myeloproliferative neoplasm; ahistiocytic disorder (e.g., a mast cell disorder or a blasticplasmacytoid dendritic cell neoplasm); a mast cell disorder, e.g.,systemic mastocytosis or mast cell leukemia; B-cell prolymphocyticleukemia, plasma cell myeloma, and “preleukemia” which are a diversecollection of hematological conditions united by ineffective production(or dysplasia) of myeloid blood cells, and the like. Further diseasesassociated with expression of CD19 expression include, but not limitedto, e.g., atypical and/or non-classical cancers, malignancies,precancerous conditions or proliferative diseases associated withexpression of CD19. Non-cancer related indications associated withexpression of CD19 include, but are not limited to, e.g., autoimmunedisease, (e.g., lupus), inflammatory disorders (allergy and asthma) andtransplantation. In some embodiments, the tumor antigen-expressing cellsexpress, or at any time expressed, mRNA encoding the tumor antigen. Inan embodiment, the tumor antigen-expressing cells produce the tumorantigen protein (e.g., wild-type or mutant), and the tumor antigenprotein may be present at normal levels or reduced levels. In anembodiment, the tumor antigen-expressing cells produced detectablelevels of a tumor antigen protein at one point, and subsequentlyproduced substantially no detectable tumor antigen protein. In otherembodiments, the disease is a CD19-negative cancer, e.g., aCD19-negative relapsed cancer. In some embodiments, the tumor antigen(e.g., CD19)-expressing cell expresses, or at any time expressed, mRNAencoding the tumor antigen. In an embodiment, the tumor antigen (e.g.,CD19)-expressing cell produces the tumor antigen protein (e.g.,wild-type or mutant), and the tumor antigen protein may be present atnormal levels or reduced levels. In an embodiment, the tumor antigen(e.g., CD19)-expressing cell produced detectable levels of a tumorantigen protein at one point, and subsequently produced substantially nodetectable tumor antigen protein.

The term “costimulatory molecule” refers to the cognate binding partneron a T cell that specifically binds with a costimulatory ligand, therebymediating a costimulatory response by the T cell, such as, but notlimited to, proliferation. Costimulatory molecules are cell surfacemolecules other than antigen receptors or their ligands that arerequired for an efficient immune response. Costimulatory moleculesinclude, but are not limited to MHC class I molecule, TNF receptorproteins, Immunoglobulin-like proteins, cytokine receptors, integrins,signalling lymphocytic activation molecules (SLAM proteins), activatingNK cell receptors, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27,CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3,CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2,SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha,CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4,IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL,CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18,LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4(CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160(BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, CD19a, and a ligand that specifically binds with CD83.

A costimulatory intracellular signaling domain refers to anintracellular portion of a costimulatory molecule. The intracellularsignaling domain can comprise the entire intracellular portion, or theentire native intracellular signaling domain, of the molecule from whichit is derived, or a functional fragment thereof.

As used herein, a “value of responder or relapser status” includes ameasure (e.g., level) predictive of responsiveness or relapse of asubject to a treatment (e.g., a treatment that comprises, or consistsof, a CAR-expressing cell therapy as described herein). In someembodiments, the measure is qualitative or quantitative. In someembodiments, the value of responder or relapser status is completeresponder, partial responder, non-responder, relapser or non-relapser.In some embodiments, the value of responder or relapser status is aprobability of being a complete responder, a partial responder, anon-responder, a relapser or a non-relapser. In some embodiments, thevalue of responder or relapser status can be determined based on themeasure of any of (i)-(viii) as described herein.

With respect to responsiveness, a subject responds to treatment if aparameter of a cancer (e.g., a hematological cancer, e.g., cancer cellgrowth, proliferation and/or survival) in the subject is retarded orreduced by a detectable amount, e.g., about 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% or more as determined by any appropriate measure,e.g., by mass, cell count or volume. In one example, a subject respondsto treatment if the subject experiences a life expectancy extended byabout 5%, 10%, 20%, 30%, 40%, 50% or more beyond the life expectancypredicted if no treatment is administered. In another example, a subjectresponds to treatment, if the subject has an increased disease-freesurvival, overall survival or increased time to progression.

Several methods can be used to determine if a patient responds to atreatment including, for example, criteria provided by NCCN ClinicalPractice Guidelines in Oncology (NCCN Guidelines®). For example, in thecontext of B-ALL, a complete response or complete responder, may involveone or more of: <5% BM blast, >1000 neutrophil/ANC (/μL). >100,000platelets (/μL) with no circulating blasts or extramedullary disease (Nolymphadenopathy, splenomegaly, skin/gum infiltration/testicular mass/CNSinvolvement), Trilineage hematopoiesis, and no recurrence for 4 weeks. Apartial responder may involve one or more of ≥50% reduction in BMblast, >1000 neutrophil/ANC (/μL). >100,000 platelets (/μL). Anon-responder can show disease progression, e.g., >25% in BM blasts.

A “complete responder” as used herein refers to a subject having adisease, e.g., a cancer, who exhibits a complete response, e.g., acomplete remission, to a treatment. A complete response may beidentified, e.g., using the NCCN Guidelines®, or Cheson et al, J ClinOncol 17:1244 (1999) and Cheson et al., “Revised Response Criteria forMalignant Lymphoma”, J Clin Oncol 25:579-586 (2007) (both of which areincorporated by reference herein in their entireties), as describedherein.

A “partial responder” as used herein refers to a subject having adisease, e.g., a cancer, who exhibits a partial response, e.g., apartial remission, to a treatment. A partial response may be identified,e.g., using the NCCN Guidelines®, or Cheson criteria as describedherein.

A “non-responder” as used herein refers to a subject having a disease,e.g., a cancer, who does not exhibit a response to a treatment, e.g.,the patient has stable disease or progressive disease. A non-respondermay be identified, e.g., using the NCCN Guidelines®, or Cheson criteriaas described herein.

The term “relapse” as used herein refers to reappearance of a disease(e.g., cancer) after an initial period of responsiveness, e.g., afterprior treatment with a therapy, e.g., cancer therapy (e.g., completeresponse or partial response). The initial period of responsiveness mayinvolve the level of cancer cells falling below a certain threshold,e.g., below 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%. The reappearance mayinvolve the level of cancer cells rising above a certain threshold,e.g., above 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%. For example, e.g., inthe context of B-ALL, the reappearance may involve, e.g., a reappearanceof blasts in the blood, bone marrow (>5%), or any extramedullary site,after a complete response. A complete response, in this context, mayinvolve <5% BM blast. More generally, in an embodiment, a response(e.g., complete response or partial response) can involve the absence ofdetectable MRD (minimal residual disease). In an embodiment, the initialperiod of responsiveness lasts at least 1, 2, 3, 4, 5, or 6 days; atleast 1, 2, 3, or 4 weeks; at least 1, 2, 3, 4, 6, 8, 10, or 12 months;or at least 1, 2, 3, 4, or 5 years.

In some embodiments, a therapy that includes a CD19 inhibitor, e.g., aCD19 CAR therapy, may relapse or be refractory to treatment. The relapseor resistance can be caused by CD19 loss (e.g., an antigen lossmutation) or other CD19 alteration that reduces the level of CD19 (e.g.,caused by clonal selection of CD19-negative clones). A cancer thatharbors such CD19 loss or alteration is referred to herein as a“CD19-negative cancer” or a “CD19-negative relapsed cancer”). It shallbe understood that a CD19-negative cancer need not have 100% loss ofCD19, but a sufficient reduction to reduce the effectiveness of a CD19therapy such that the cancer relapses or becomes refractory. In someembodiments, a CD19-negative cancer results from a CD19 CAR therapy. Insome embodiments, a CD19-negative multiple myeloma can be treated with aCD19 CAR-expressing therapy, e.g., as described in PCT/US2015/024671,filed Apr. 7, 2015 (e.g., paragraphs 9 and 90, and Example 6 therein),which is incorporated by reference in its entirety. In some embodiments,a CD19-negative cancer can be treated with a CAR-expressing therapy,e.g., a CD123 CAR-expressing therapy, e.g., as described inPCT/US2015/045898 filed Aug. 19, 2015 (e.g., p. 26, p. 30, and Example 7therein) which is incorporated by reference in its entirety.

The term “endogenous” refers to any material from or produced inside anorganism, cell, tissue or system.

The term “effective amount” or “therapeutically effective amount” areused interchangeably herein, and refer to an amount of a compound,formulation, material, or composition, as described herein effective toachieve a particular biological result.

The term “exogenous” refers to any material introduced from or producedoutside an organism, cell, tissue or system.

The term “expression” refers to the transcription and/or translation ofa particular nucleotide sequence driven by a promoter.

The term “flexible polypeptide linker” or “linker” as used in thecontext of a scFv refers to a peptide linker that consists of aminoacids such as glycine and/or serine residues used alone or incombination, to link variable heavy and variable light chain regionstogether. In one embodiment, the flexible polypeptide linker is aGly/Ser linker and comprises the amino acid sequence(Gly-Gly-Gly-Ser)_(n), where n is a positive integer equal to or greaterthan 1. For example, n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9 andn=10 (SEQ ID NO:28). In one embodiment, the flexible polypeptide linkersinclude, but are not limited to, (Gly₄Ser)₄ (SEQ ID NO:29) or (Gly₄Ser)₃ (SEQ ID NO:30). In another embodiment, the linkers includemultiple repeats of (Gly₂Ser), (GlySer) or (Gly₃Ser) (SEQ ID NO:31).Also included within the scope of the invention are linkers described inWO2012/138475, incorporated herein by reference.

The terms “homology” or “identity,” as used interchangeably herein,refer to sequence similarity between two polynucleotide sequences orbetween two polypeptide sequences, with identity being a more strictcomparison. The phrases “percent identity or homology” and “% identityor homology” refer to the percentage of sequence similarity found in acomparison of two or more polynucleotide sequences or two or morepolypeptide sequences. “Sequence similarity” refers to the percentsimilarity in base pair sequence (as determined by any suitable method)between two or more polynucleotide sequences. Two or more sequences canbe anywhere from 0-100% similar, or any integer value there between.Identity or similarity can be determined by comparing a position in eachsequence that can be aligned for purposes of comparison. When a positionin the compared sequence is occupied by the same nucleotide base oramino acid, then the molecules are identical at that position. A degreeof similarity or identity between polynucleotide sequences is a functionof the number of identical or matching nucleotides at positions sharedby the polynucleotide sequences. A degree of identity of polypeptidesequences is a function of the number of identical amino acids atpositions shared by the polypeptide sequences. A degree of homology orsimilarity of polypeptide sequences is a function of the number of aminoacids at positions shared by the polypeptide sequences. The term“substantial homology,” as used herein, refers to homology of at least50%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95% or more.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab, F(ab)2 or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies and antibody fragments thereofare human immunoglobulins (recipient antibody or antibody fragment) inwhich residues from a complementary-determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity, and capacity. In some instances, Fv frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, a humanizedantibody/antibody fragment can comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. These modifications can further refine and optimize antibodyor antibody fragment performance. In general, the humanized antibody orantibody fragment thereof will comprise substantially all of at leastone, and typically two, variable domains, in which all or substantiallyall of the CDR regions correspond to those of a non-human immunoglobulinand all or a significant portion of the FR regions are those of a humanimmunoglobulin sequence. The humanized antibody or antibody fragment canalso comprise at least a portion of an immunoglobulin constant region(Fc), typically that of a human immunoglobulin. For further details, seeJones et al., NATURE, 321: 522-525, 1986; Reichmann et al., NATURE, 332:323-329, 1988; Presta, CURR. OP. STRUCT. BIOL., 2: 593-596, 1992.

“Immune effector cell,” as that term is used herein, refers to a cellthat is involved in an immune response, e.g., in the promotion of animmune effector response. Examples of immune effector cells include Tcells, e.g., alpha/beta T cells and gamma/delta T cells, B cells,natural killer (NK) cells, natural killer T (NK-T) cells, mast cells,and myeloid-derived phagocytes.

“Immune effector function or immune effector response,” as that term isused herein, refers to function or response, e.g., of an immune effectorcell, that enhances or promotes an immune attack of a target cell. E.g.,an immune effector function or response refers a property of a T or NKcell that promotes killing or the inhibition of growth or proliferation,of a target cell. In the case of a T cell, primary stimulation andco-stimulation are examples of immune effector function or response.

The term “effector function” refers to a specialized function of a cell.Effector function of a T cell, for example, may be cytolytic activity orhelper activity including the secretion of cytokines.

The term “4-1BB” refers to a member of the TNFR superfamily with anamino acid sequence provided as GenBank Acc. No. AAA62478.2, or theequivalent residues from a non-human species, e.g., mouse, rodent,monkey, ape and the like; and a “4-1BB costimulatory domain” is definedas amino acid residues 214-255 of GenBank Acc No. AAA62478.2, or theequivalent residues from a non-human species, e.g., mouse, rodent,monkey, ape and the like. In one aspect, the “4-1BB costimulatorydomain” is the sequence provided as SEQ ID NO:14 or the equivalentresidues from a non-human species, e.g., mouse, rodent, monkey, ape andthe like.

An “intracellular signaling domain,” as the term is used herein, refersto an intracellular portion of a molecule. The intracellular signalingdomain can generate a signal that promotes an immune effector functionof the CAR containing cell, e.g., a CAR-expressing cell, e.g., a T cellor an NK cell. Examples of immune effector function, e.g., in aCAR-expressing cell include, cytolytic activity and helper activity,including the secretion of cytokines. In embodiments, the intracellularsignal domain transduces the effector function signal and directs thecell to perform a specialized function. While the entire intracellularsignaling domain can be employed, in many cases it is not necessary touse the entire chain. To the extent that a truncated portion of theintracellular signaling domain is used, such truncated portion may beused in place of the intact chain as long as it transduces the effectorfunction signal. The term intracellular signaling domain is thus meantto include any truncated portion of the intracellular signaling domainsufficient to transduce the effector function signal.

In an embodiment, the intracellular signaling domain can comprise aprimary intracellular signaling domain. Exemplary primary intracellularsignaling domains include those derived from the molecules responsiblefor primary stimulation, or antigen dependent simulation. In anembodiment, the intracellular signaling domain can comprise acostimulatory intracellular domain. Exemplary costimulatoryintracellular signaling domains include those derived from moleculesresponsible for costimulatory signals, or antigen independentstimulation. For example, in the case of a CAR-expressing cell (e.g., aT cell, an NK cell), a primary intracellular signaling domain cancomprise a cytoplasmic sequence of a T cell receptor, and acostimulatory intracellular signaling domain can comprise cytoplasmicsequence from co-receptor or costimulatory molecule.

A primary intracellular signaling domain can comprise a signaling motifwhich is known as an immunoreceptor tyrosine-based activation motif orITAM. Examples of ITAM containing primary cytoplasmic signalingsequences include, but are not limited to, those derived from CD3 zeta,FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22,CD79a, CD79b, CD278 (“ICOS”), FccRI, CD66d, CD32, DAP10, and DAP12.

As used herein, “in vitro transcribed RNA” refers to RNA, preferablymRNA, that has been synthesized in vitro. Generally, the in vitrotranscribed RNA is generated from an in vitro transcription vector. Thein vitro transcription vector comprises a template that is used togenerate the in vitro transcribed RNA.

The term “isolated” means altered or removed from the natural state. Forexample, a nucleic acid or a peptide naturally present in a livinganimal is not “isolated,” but the same nucleic acid or peptide partiallyor completely separated from the coexisting materials of its naturalstate is “isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

The term “lentivirus” refers to a genus of the Retroviridae family.Lentiviruses are unique among the retroviruses in being able to infectnon-dividing cells; they can deliver a significant amount of geneticinformation into the DNA of the host cell, so they are one of the mostefficient methods of a gene delivery vector. HIV, SIV, and FIV are allexamples of lentiviruses.

The term “lentiviral vector” refers to a vector derived from at least aportion of a lentivirus genome, including especially a self-inactivatinglentiviral vector as provided in Milone et al., MOL. THER. 17(8):1453-1464 (2009). Other examples of lentivirus vectors that may be usedin the clinic, include but are not limited to, e.g., the LENTIVECTOR®gene delivery technology from Oxford BioMedica, the LENTIMAX™ vectorsystem from Lentigen and the like. Nonclinical types of lentiviralvectors are also available and would be known to one skilled in the art.

The term ‘low, immune enhancing, dose” when used in conjunction with anmTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., RAD001 orrapamycin, or a catalytic mTOR inhibitor, refers to a dose of mTORinhibitor that partially, but not fully, inhibits mTOR activity, e.g.,as measured by the inhibition of P70 S6 kinase activity. Methods forevaluating mTOR activity, e.g., by inhibition of P70 S6 kinase, arediscussed herein. The dose is insufficient to result in complete immunesuppression but is sufficient to enhance the immune response. In anembodiment, the low, immune enhancing, dose of mTOR inhibitor results ina decrease in the number of PD-1 positive immune effector cells, e.g., Tcells or NK cells and/or an increase in the number of PD-1 negativeimmune effector cells, e.g., T cells or NK cells, or an increase in theratio of PD-1 negative immune effector cells, e.g., T cells or NKcells/PD-1 positive immune effector cells, e.g., T cells or NK cells.

In general, the term “naïve T cell” refers to immune cells that compriseantigen-inexperienced cells, e.g., immune cells that are precursors ofmemory cells. In some embodiments, naïve T cells may be differentiated,but have not yet encountered their cognate antigen, and therefore areactivated T cells or memory T cells. In some embodiments, naïve T cellsmay be characterized by expression of CD62L, CD27, CCR7, CD45RA, CD28,and CD127, and the absence of CD95, or CD45RO isoform. In certainembodiments, a naïve T cells is a type of younger T cell as describedherein.

The term “less exhausted” or “less exhausted phenotype” refers to immuneeffector cells that have reduced (e.g., lack) expression of immune cellexhaustion markers, e.g. PD1, TIM3, and LAG3. In some embodiments, aless exhausted cell may be a younger T cell as described herein.

The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleicacid (DNA) or ribonucleic acid (RNA), or a combination of a DNA or RNAthereof, and polymers thereof in either single- or double-stranded form.The term “nucleic acid” includes a gene, CDNA or an mRNA. In oneembodiment, the nucleic acid molecule is synthetic (e.g., chemicallysynthesized) or recombinant. Unless specifically limited, the termencompasses nucleic acids containing analogues or derivatives of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), alleles,orthologs, SNPs, and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., NUCLEIC ACID RES. 19:5081 (1991);Ohtsuka et al., J. BIOL. CHEM. 260:2605-2608 (1985); and Rossolini etal., MOL. CELL. Probes 8:91-98 (1994)). In the context of the presentinvention, the following abbreviations for the commonly occurringnucleic acid bases are used. “A” refers to adenosine, “C” refers tocytosine, “G” refers to guanosine, “T” refers to thymidine, and “U”refers to uridine.

A “nucleic acid” “marker” or “biomarker” is a nucleic acid (e.g., DNA,mRNA, CDNA) encoded by or corresponding to a marker as described herein.For example, such marker nucleic acid molecules include DNA (e.g.,genomic DNA and CDNA) comprising the entire or a partial sequence of anyof the nucleic acid sequences set forth, or the complement orhybridizing fragment of such a sequence. The marker nucleic acidmolecules also include RNA comprising the entire or a partial sequenceof any of the nucleic acid sequences set forth herein, or the complementof such a sequence, wherein all thymidine residues are replaced withuridine residues. A “marker protein” is a protein encoded by orcorresponding to a marker of the invention. A marker protein comprisesthe entire or a partial sequence of a protein encoded by any of thesequences set forth herein, or a fragment thereof. The terms “protein”and “polypeptide” are used interchangeably herein.

An “overexpression” or “significantly higher level of expression” of thegene products refers to an expression level or copy number in a testsample that is greater than the standard error of the assay employed toassess the level of expression. In embodiments, the overexpression canbe at least two, at least three, at least four, at least five, or atleast ten or more times the expression level of the gene in a controlsample or the average expression level of gene products in severalcontrol samples.

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. A polypeptide includes a natural peptide, arecombinant peptide, or a combination thereof.

As used herein, a “poly(A)” is a series of adenosines attached bypolyadenylation to the mRNA. In some embodiments of a construct fortransient expression, the polyA is between 50 and 5000 (SEQ ID NO: 34)(e.g., 2000; SEQ ID NO: 32), e.g., 64 (SEQ ID NO: 37), e.g., greaterthan 100 (e.g., 150, SEQ ID NO: 33), e.g., greater than 400 (SEQ ID NO:38). poly(A) sequences can be modified chemically or enzymatically tomodulate mRNA functionality such as localization, stability orefficiency of translation.

The term “probe” refers to any molecule which is capable of selectivelybinding to a specifically intended target molecule, for example a markerof the invention. Probes can be either synthesized by one skilled in theart, or derived from appropriate biological preparations. For purposesof detection of the target molecule, probes can be specifically designedto be labeled, as described herein. Examples of molecules that can beutilized as probes include, but are not limited to, RNA, DNA, proteins,antibodies, and organic monomers.

The term “promoter” refers to a DNA sequence recognized by the syntheticmachinery of the cell, or introduced synthetic machinery, required toinitiate the specific transcription of a polynucleotide sequence.

The term “promoter/regulatory sequence” refers to a nucleic acidsequence which is required for expression of a gene product operablylinked to the promoter/regulatory sequence. In some instances, thissequence may be the core promoter sequence and in other instances, thissequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

The term “prophylaxis” as used herein means the prevention of orprotective treatment for a disease or disease state.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Asanother example, a range such as 95-99% identity, includes somethingwith 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This appliesregardless of the breadth of the range.

The term “recombinant antibody” refers to an antibody which is generatedusing recombinant DNA technology, such as, for example, an antibodyexpressed by a bacteriophage or yeast expression system. The term shouldalso be construed to mean an antibody which has been generated by thesynthesis of a DNA molecule encoding the antibody and which DNA moleculeexpresses an antibody protein, or an amino acid sequence specifying theantibody, wherein the DNA or amino acid sequence has been obtained usingrecombinant DNA or amino acid sequence technology which is available andwell known in the art.

“Refractory” as used herein refers to a disease, e.g., cancer, that doesnot respond to a treatment. In embodiments, a refractory cancer can beresistant to a treatment before or at the beginning of the treatment. Inother embodiments, the refractory cancer can become resistant during atreatment. A refractory cancer is also called a resistant cancer.

In embodiments, a reference or control level or activity is the leveland/or activity in a subject, e.g., a sample obtained from one or moreof: a baseline or prior value for the subject (e.g., prior to treatmentwith a CAR-expressing cell); the subject at a different time interval;an average or median value for a cancer patient population; a healthycontrol; or a healthy subject population (e.g., a control).

“Sample,” “tissue sample,” “patient sample,” “patient cell or tissuesample” or “specimen” each refers to a biological sample obtained from atissue or bodily fluid of a subject or patient. The source of the tissuesample can be solid tissue as from a fresh, frozen and/or preservedorgan, tissue sample, biopsy, or aspirate; blood or any bloodconstituents (e.g., serum, plasma); bodily fluids such as urine,cerebral spinal fluid, whole blood, plasma and serum. The sample caninclude a non-cellular fraction (e.g., urine, plasma, serum, or othernon-cellular body fluid). In one embodiment, the sample is a urinesample. In other embodiments, the body fluid from which the sample isobtained from an individual comprises blood (e.g., whole blood). In anembodiment, the sample is a whole blood sample obtained from thesubject. In certain embodiments, the blood can be further processed toobtain plasma or serum. In an embodiment, the sample is an apheresissample obtained from the blood of the subject. In an embodiment, thesample is a manufactured product sample, e.g., genetically engineered Tcells obtained from the blood of the subject, e.g., a manufacturedCAR-expressing cell (e.g., T cell, NK cell) product, e.g., amanufactured CD19 CAR-expressing cell product. In another embodiment,the sample contains a tissue, cells (e.g., peripheral blood mononuclearcells (PBMC)). For example, the sample can be a fine needle biopsysample, an archival sample (e.g., an archived sample with a knowndiagnosis and/or treatment history), a histological section (e.g., afrozen or formalin-fixed section, e.g., after long term storage), amongothers. The term sample includes any material obtained and/or derivedfrom a biological sample, including a polypeptide, and nucleic acid(e.g., genomic DNA, CDNA, RNA) purified or processed from the sample.Purification and/or processing of the sample can involve one or more ofextraction, concentration, antibody isolation, sorting, concentration,fixation, addition of reagents and the like. The sample can containcompounds that are not naturally intermixed with the tissue in naturesuch as preservatives, anticoagulants, buffers, fixatives, nutrients,antibiotics or the like.

The term “product” or “manufactured product” as used herein, refers to amanufactured composition comprising a genetically engineered cell (e.g.,an immune effector cell), e.g., a population of cells in which aplurality of cells are engineered to express a CAR, e.g., a CARdescribed herein. A manufactured product can be any geneticallyengineered immune effector cell (e.g., T cell, NK cell), e.g.,genetically engineered immune effector cells obtained from the blood ofthe subject, e.g., a manufactured CAR-expressing cell product, e.g., amanufactured CD19 CAR-expressing cell product. In an embodiment, a cell(e.g., an immune effector cell) engineered to express a CAR may beobtained from an activated cryopreserved expanded cell population (e.g.,an expanded immune effector cell population).

The term “signaling domain” refers to the functional portion of aprotein which acts by transmitting information within the cell toregulate cellular activity via defined signaling pathways by generatingsecond messengers or functioning as effectors by responding to suchmessengers.

The amount of a biomarker, e.g., expression of gene products (e.g., oneor more the biomarkers described herein), in a subject is“significantly” higher or lower than the normal amount of a marker, ifthe amount of the marker is greater or less, respectively, than thenormal level by an amount greater than the standard error of the assayemployed to assess amount, or at least two, three, four, five, ten ormore times that amount. Alternatively, the amount of the marker in thesubject can be considered “significantly” higher or lower than thenormal amount if the amount is at least about 1.5, two, at least aboutthree, at least about four, or at least about five times, higher orlower, respectively, than the normal amount of the marker.

The term “specifically binds,” refers to an antibody, or a ligand, whichrecognizes and binds with a cognate binding partner protein present in asample, but which antibody or ligand does not substantially recognize orbind other molecules in the sample.

The term “stimulation,” refers to a primary response induced by bindingof a stimulatory molecule (e.g., a TCR/CD3 complex) with its cognateligand thereby mediating a signal transduction event, such as, but notlimited to, signal transduction via the TCR/CD3 complex. Stimulation canmediate altered expression of certain molecules, such as down regulationof TGF-β, and/or reorganization of cytoskeletal structures, and thelike.

The term “stimulatory molecule,” refers to a molecule expressed by a Tcell that provides the primary cytoplasmic signaling sequence(s) thatregulate primary activation of the TCR complex in a stimulatory way forat least some aspect of the T cell signaling pathway. In one aspect, theprimary signal is initiated by, for instance, binding of a TCR/CD3complex with an MHC molecule loaded with peptide, and which leads tomediation of a T cell response, including, but not limited to,proliferation, activation, differentiation, and the like. A primarycytoplasmic signaling sequence (also referred to as a “primary signalingdomain”) that acts in a stimulatory manner may contain a signaling motifwhich is known as immunoreceptor tyrosine-based activation motif orITAM. Examples of an ITAM containing cytoplasmic signaling sequence thatis of particular use in the invention includes, but is not limited to,those derived from CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa,FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a,CD79b, DAP10, and DAP12. In a specific CAR of the invention, theintracellular signaling domain in any one or more CARS of the inventioncomprises an intracellular signaling sequence, e.g., a primary signalingsequence of CD3-zeta. In a specific CAR of the invention, the primarysignaling sequence of CD3-zeta is the sequence provided as SEQ ID NO:18(mutant CD3 zeta), or the equivalent residues from a non-human species,e.g., mouse, rodent, monkey, ape and the like. In a specific CAR of theinvention, the primary signaling sequence of CD3-zeta is the sequence asprovided in SEQ ID NO:20 (wild-type human CD3 zeta), or the equivalentresidues from a non-human species, e.g., mouse, rodent, monkey, ape andthe like.

The term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals, human). In anembodiment, a subject is a mammal. In an embodiment, a subject is ahuman. In an embodiment, a subject is a patient.

The term “therapeutic” as used herein means a treatment. A therapeuticeffect is obtained by reduction, suppression, remission, or eradicationof a disease state.

The term “transfected” or “transformed” or “transduced” refers to aprocess by which exogenous nucleic acid is transferred or introducedinto the host cell. A “transfected” or “transformed” or “transduced”cell is one which has been transfected, transformed or transduced withexogenous nucleic acid. The cell includes the primary subject cell andits progeny.

As used herein, “transient” refers to expression of a non-integratedtransgene for a period of hours, days or weeks, wherein the period oftime of expression is less than the period of time for expression of thegene if integrated into the genome or contained within a stable plasmidreplicon in the host cell.

The term “transmembrane domain,” refers to a polypeptide that spans theplasma membrane. In an embodiment, it links an extracellular sequence,e.g., a switch domain, an extracellular recognition element, e.g., anantigen binding domain, an inhibitory counter ligand binding domain, orcostimulatory ECD domain, to an intracellular sequence, e.g., to aswitch domain or an intracellular signaling domain. A transmembranedomain can include one or more additional amino acids adjacent to thetransmembrane region, e.g., one or more amino acid associated with theextracellular region of the protein from which the transmembrane wasderived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of theextracellular region) and/or one or more additional amino acidsassociated with the intracellular region of the protein from which thetransmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 upto 15 amino acids of the intracellular region). Examples oftransmembrane domains are disclosed herein.

The terms “treat”, “treatment” and “treating” refer to the reduction oramelioration of the progression, severity and/or duration of aproliferative disorder, or the amelioration of one or more symptoms(e.g., one or more discernible symptoms) of a proliferative disorderresulting from the administration of one or more therapies (e.g., one ormore therapeutic agents such as a CAR of the invention). In specificembodiments, the terms “treat”, “treatment” and “treating” refer to theamelioration of at least one measurable physical parameter of aproliferative disorder, such as growth of a tumor, not necessarilydiscernible by the patient. In other embodiments the terms “treat”,“treatment” and “treating”-refer to the inhibition of the progression ofa proliferative disorder, either physically by, e.g., stabilization of adiscernible symptom, physiologically by, e.g., stabilization of aphysical parameter, or both. In other embodiments the terms “treat”,“treatment” and “treating” refer to the reduction or stabilization oftumor size or cancerous cell count.

An “underexpression” or “significantly lower level of expression” ofproducts (e.g., the markers set forth herein) refers to an expressionlevel in a test sample that is greater than the standard error of theassay employed to assess expression, for example, at least 1.5, twice,at least three, at least four, at least five, or at least ten or moretimes less than the expression level of the gene in a control sample, orthe average expression level of gene products in several controlsamples.

The term “xenogeneic” refers to a graft derived from an animal of adifferent species.

As used herein, the term “young T cell” or “younger T cell”, refers toan immune effector cell that comprises a less differentiated phenotype,e.g., a younger cell, e.g., a young T cell. In some embodiments, ayounger T cell may be a naïve T cell (T_(N)). In some embodiments, ayoung T cell may be characterized by expression of CD62L, and theabsence of CD25, CD44, or CD45RO isoform. In some embodiments, a youngerT cell may be a memory stem cell (T_(SCM)). In some embodiments, ayounger T cells may be a central memory T cell (T_(CM)). Phenotypicmarkers associated with T_(N), T_(SCM) and T_(CM) are disclosed in,e.g., Maus, M. et al. (2014) Annu. Rev. Immunol. 32:189-225 (see forexample, FIG. 3), incorporated by reference herein. Exemplary phenotypesof T_(N) include one or more (or all) of the following: CD45RA+,CD45RO−, CD62L^(high), CCR7^(high), CD95−, CD122−, CD27^(high), CD28+,CD57−, KLRG-1−, or long telomere length (or any combination of two,three, four, five, six, seven, eight, nine, or all of the aforesaidT_(N) markers). Exemplary phenotypes of T_(SCM) include one or more (orall) of the following: CD45RA+, CD45RO−, CD62L^(high), CCR7^(high),CD95+, CD122+, CD27^(high), CD28^(high), CD57−, KLRG-1−, or longtelomere length (or any combination of two, three, four, five, six,seven, eight, nine, or all of the aforesaid T_(SCM) markers). Exemplaryphenotypes of T_(CM) include one or more (or all) of the following:CD45RA−, CD45RO^(high), CD62L^(high), CCR7+, CD95+, CD122^(high), CD27+,CD28^(high), CD57−, KLRG-1−/+, or long/intermediate telomere length (orany combination of two, three, four, five, six, seven, eight, nine, orall of the aforesaid T_(CM) markers).

As used herein, the term “older T cell” refers to an immune effectorcell that comprises a more exhausted phenotype. In some embodiments, anolder T cell may be an effector memory T cell (T_(EM)). In otherembodiments, an older T cell may be an effector T cell (T_(EFF)). Inother embodiments, an older T cell has an exhausted phenotype.Phenotypic markers associated with T_(EM), T_(EFF) and exhausted T cellsare disclosed in, e.g., Maus, M. et al. (2014) Annu. Rev. Immunol.32:189-225 (see for example, FIG. 3), incorporated by reference herein.Exemplary phenotypes of T_(EM) include one or more (or all) of thefollowing: CD45RA−/+, CD45RO^(high), CD62L−, CCR7−, CD95−, CD122^(high),CD27−/+, CD28−/+, CD57^(low), KLRG-1+, or intermediate telomere length(or any combination of two, three, four, five, six, seven, eight, nine,or all of the aforesaid T_(EM) markers). Exemplary phenotypes of T_(EFF)include one or more (or all) of the following: CD45RA−/+, CD45RO+,CD62L−, CCR7−, CD95^(high), CD122−/+, CD27−, CD28−, CD57+,KLRG-1^(high), or short/intermediate telomere length (or any combinationof two, three, four, five, six, seven, eight, nine, or all of theaforesaid T_(EFF) markers). Exemplary phenotypes of an exhausted T cellphenotype include one or more (or all) of the following: CD45RA−/+,CD45RO+, CD62L−, CCR7-, CD95^(high), CD122^(low), CD27−, CD28−,CD57^(high), KLRG-1^(high), or short telomere length (or any combinationof two, three, four, five, six, seven, eight, nine, or all of theaforesaid markers).

The term “zeta” or alternatively “zeta chain”, “CD3-zeta” or “TCR-zeta”is defined as the protein provided as GenBank Acc. No. BAG36664.1, orthe equivalent residues from a non-human species, e.g., mouse, rodent,monkey, ape and the like, and a “zeta stimulatory domain” oralternatively a “CD3-zeta stimulatory domain” or a “TCR-zeta stimulatorydomain” is defined as the amino acid residues from the cytoplasmicdomain of the zeta chain that are sufficient to functionally transmit aninitial signal necessary for T cell activation. In one aspect thecytoplasmic domain of zeta comprises residues 52 through 164 of GenBankAcc. No. BAG36664.1 or the equivalent residues from a non-human species,e.g., mouse, rodent, monkey, ape and the like, that are functionalorthologs thereof. In one aspect, the “zeta stimulatory domain” or a“CD3-zeta stimulatory domain” is the sequence provided as SEQ ID NO:18.In one aspect, the “zeta stimulatory domain” or a “CD3-zeta stimulatorydomain” is the sequence provided as SEQ ID NO:20.

Various aspects of the invention are described in further detail below.Additional definitions are set out throughout the specification.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Biomarkers predicting response to a therapy in subjects having cancer(e.g., a hematological cancer such as chronic lymphocytic leukemia (CLL)and acute lymphoblastic leukemia (ALL)) are provided herein.

In one aspect, biomarkers predicting response to a cell expressing aCAR, e.g., a CD19 CAR-expressing cell (e.g., T cell, NK cell) (e.g., aCD19 CAR-expressing cell described herein such as, e.g., CTL019) insubjects having CLL and ALL are provided herein.

Methods are provided for the diagnosis and monitoring of treatment ofcancer (e.g., a hematological cancer such as ALL and CLL) based ondetection of certain biomarkers in samples from patients who have, orare suspected of having, cancer. Further, expression of one or more suchbiomarkers can be used to distinguish subjects that respond favorably toa CAR-expressing cell (e.g., T cell, NK cell) therapy (e.g., “completeresponders” or “CR”) from subjects that don't respond to aCAR-expressing cell therapy (e.g., “non-responders” or “NR”) and fromsubjects that have a partial response to a CAR-expressing cell therapy(e.g., “partial responders” or “PR”).

Use of Biomarkers to Evaluate Disease Progression and Predict SubjectResponse to CAR-Expressing Cell Therapy

In an embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more genes inTable 1A, Table 1B, Table 7A, Table 7B, Table 8, Table 9, Table 10,Table 14, Table 15, Table 16 (e.g., CCL20, IL-17a and/or IL-6), Table17, Table 18, Table 20, PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L,KLRG1 and/or a CD19 CAR-expressing cell (e.g., T cell, NK cell) genesignature can be used with methods of the present disclosure to acquirea disease progression value. The disease progression value can be usedfor, e.g., in evaluating the effectiveness of therapies in treatingcancer (e.g., a hematological cancer such as ALL and CLL). In anembodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more genes in Table1A, Table 1B, Table 7A, Table 7B, Table 8, Table 9, Table 10, Table 14,Table 15, Table 16 (e.g., CCL20, IL-17a and/or IL-6), Table 17, Table18, Table 20, PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L, and/or KLRG1and/or a CD19 CAR-expressing cell gene signature are used to classify asubject as a complete responder, partial responder, or non-responder toCAR-expressing cell therapy (e.g., a CD19 CAR-expressing cell therapydescribed herein such as, e.g., CTL019). In an embodiment, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20 or more genes in Table 1A, Table 1B, Table 7A,Table 7B, Table 8, Table 9, Table 10, Table 14, Table 15, Table 16(e.g., CCL20, IL-17a and/or IL-6), Table 17, Table 18, Table 20, PD-1,LAG-3, TIM-3, CD57, CD27, CD122, CD62L, and/or KLRG1, and/or a CD19CAR-expressing cell gene signature are used to predict a subject'sresponsiveness to a CAR-expressing cell therapy (e.g., a CD19CAR-expressing cell therapy described herein such as, e.g., CTL019).

Subjects

For any of the methods and kits disclosed herein, the subject treated,or the subject from which the value is obtained, is a subject having, orat risk of having, cancer at any stage of treatment. Exemplary cancersinclude, but are not limited to, B-cell acute lymphocytic leukemia(B-ALL), T-cell acute lymphocytic leukemia (T-ALL), acute lymphocyticleukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocyticleukemia (CLL), B cell promyelocytic leukemia, blastic plasmacytoiddendritic cell neoplasm, Burkitts lymphoma, diffuse large B celllymphoma, follicular lymphoma, hairy cell leukemia, small cell- or alarge cell-follicular lymphoma, malignant lymphoproliferativeconditions, MALT lymphoma, mantle cell lymphoma (MCL), marginal zonelymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome,non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma,plasmacytoid dendritic cell neoplasm, and Waldenstrom macroglobulinemia.In an embodiment, the cancer is a hematological cancer. In anembodiment, the cancer is ALL. In another embodiment, the cancer is CLL.In an embodiment, the cancer is associated with CD19 expression.

In an embodiment, the subject has received a pretreatment of anadditional therapy, e.g., a subject that has been identified as apartial responder or non-responder and subsequently has been pretreatedwith an additional therapy. In an embodiment, the subject receivespretreatment with an mTOR inhibitor. In an embodiment, the mTORinhibitor is administered at a dose or dosing schedule described herein.In one embodiment, a low, immune enhancing dose of an mTOR inhibitor isgiven to the subject prior to treatment with a CAR-expressing cell(e.g., a T cell, an NK cell). In an embodiment, administration of a low,immune enhancing, dose of an mTOR inhibitor, e.g., an allostericinhibitor, e.g., RAD001, or a catalytic inhibitor, is initiated prior toadministration of a CAR expressing cell described herein, e.g., T cells.In an embodiment, the CAR cells are administered after a sufficienttime, or sufficient dosing, of an mTOR inhibitor, such that the level ofPD1 negative immune effector cells, e.g., T cells, or the ratio of PD1negative immune effector cells, e.g., T cells/PD1 positive immuneeffector cells, e.g., T cells, has been, at least transiently,increased. In an embodiment, the cell, e.g., T cell, to be engineered toexpress a CAR, is harvested after a sufficient time, or after sufficientdosing of the low, immune enhancing, dose of an mTOR inhibitor, suchthat the level of PD1 negative immune effector cells, e.g., T cells, orthe ratio of PD1 negative immune effector cells, e.g., T cells/PD1positive immune effector cells, e.g., T cells, in the subject orharvested from the subject has been, at least transiently, increased.

In one embodiment, the subject has received a pretreatment with acheckpoint inhibitor, e.g., a checkpoint inhibitor described herein.Examples of inhibitory molecules, e.g., checkpoint molecules includePD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/orCEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86,B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHCclass I, MHC class II, GAL9, adenosine, and TGFR (e.g., TGFRbeta). Inembodiments, an inhibitory nucleic acid, e.g., an inhibitory nucleicacid, e.g., a dsRNA, e.g., an siRNA or shRNA; or e.g., an inhibitoryprotein or system, e.g., a clustered regularly interspaced shortpalindromic repeats (CRISPR), a transcription-activator like effectornuclease (TALEN), or a zinc finger endonuclease (ZFN), e.g., asdescribed herein, can be used to inhibit expression of a molecule thatmodulates or regulates, e.g., inhibits, T-cell function in theCAR-expressing cell. In an embodiment the agent is an shRNA, e.g., anshRNA described herein. In one embodiment, the inhibitor of checkpointmolecule can be, e.g., an antibody or antibody fragment that binds to acheckpoint molecule. For example, the agent can be an antibody orantibody fragment that binds to PD1, PD-L1, PD-L2 (e.g., as describedherein) or CTLA4 (e.g., ipilimumab (also referred to as MDX-010 andMDX-101, and marketed as Yervoy®; Bristol-Myers Squibb; Tremelimumab(IgG2 monoclonal antibody available from Pfizer, formerly known asticilimumab, CP-675,206)). In an embodiment, the agent is an antibody orantibody fragment that binds to TIM3, e.g., as described herein. In anembodiment, the agent is an antibody or antibody fragment that binds toLAG3, e.g., as described herein. In an embodiment, the agent is anantibody or antibody fragment that binds to CEACAM, e.g., as describedherein.

In an embodiment, the subject receives an additional therapy incombination with CAR-expressing cell (e.g., a T cell, an NK cell)therapy (e.g., a CD19 CAR-expressing cell therapy described herein suchas, e.g., CTL019). In an embodiment, the subject receives an mTORinhibitor, e.g., an mTOR inhibitor described herein, in combination withCAR-expressing cell therapy. In one embodiment, the mTOR inhibitor isadministered at a dose and/or dosing schedule described herein. In oneembodiment, the subject receives a checkpoint inhibitor, e.g., acheckpoint inhibitor described herein, in combination withCAR-expressing cell therapy. In one embodiment, the checkpoint inhibitoris administered at a dose and/or dosing schedule described herein. In anembodiment, the subject receives a kinase inhibitor, e.g., a kinaseinhibitor described herein. In one embodiment, the kinase inhibitor isadministered at a dose and/or dosing schedule described herein.

In an embodiment, the subject has been identified as a non-responder andthe subject receives a therapy other than a CAR-expressing cell therapy,e.g., a standard of care therapy for the particular cancer type. In oneembodiment, the subject receives one or more of an anti-CD20 antibody,or functional fragment thereof (e.g., ofatumumab, rituximab,obinutuzumab), an anti-CD52 antibody or functional fragment thereof(e.g., alemtuzumab), an alkylating agent (e.g., a nitrogen mustardalkylating agent such as, e.g., bendamustine HCl, chlorambucel,cyclophosphamide), a kinase inhibitor (e.g., a kinase inhibitordescribed herein such as, e.g., a BTK inhibitor described herein or aphosphonositide-3 kinase inhibitor described herein). In one embodiment,the subject receives a stem cell transplant.

Biomarkers Assessment Analysis of CTL019 Biomarkers

Analysis of levels of expression and/or activity of gene productscorrelated with a subject's response to CAR-expressing cell (e.g., Tcell, NK cell) therapy (e.g., a CD19 CAR-expressing cell therapydescribed herein such as, e.g., CTL019) and cancer disease progression(e.g., a hematological cancer such as CLL and ALL) has led to theidentification of novel CD19 CAR-expressing cell gene signatures. Forexample, the present invention provides methods for evaluation ofexpression level of one, two, three, four, five, six, seven, eight,nine, ten, fifteen, twenty, twenty-five, thirty, thirty-five, forty,forty-five, fifty, one hundred, or more genes from Table 1A, Table 1B,Table 7A, Table 7B, Table 8, Table 9, Table 10, Table 14, Table 15,Table 16 (e.g., CCL20, IL-17a and/or IL-6), Table 17, Table 18, Table20, PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L, and KLRG1 thatcomprise a CD19 CAR-expressing cell gene signature.

Table 1A, Table 1B, Table 7A, Table 7B, Table 8, Table 9, Table 10,Table 14, Table 15, Table 16 (e.g., CCL20, IL-17a and/or IL-6), Table17, Table 18, Table 20, PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L,and/or KLRG1 lists genes (e.g., protein biomarkers) that aredifferentially expressed by complete responders compared to partialresponders and non-responders.

In some embodiments, methods of the present disclosure can be used todetermine the responsiveness of a subject to treatment with aCAR-expressing cell therapy (e.g., a CD19 CAR-expressing cell (e.g., Tcell, NK cell) therapy described herein such as, e.g., CTL019), whereina statistically significant difference in the amount, e.g., expression,and/or activity of a marker disclosed herein relative to a reference,e.g., a median value for a cancer patient population (e.g., ahematological cancer such as CLL and ALL), a median value for apopulation of healthy, cancer-free subjects, a median value for apopulation of non-responders or partial responders, in a subjectssample, then the more likely the disease is to respond to CAR-expressingcell therapy.

In an embodiment, the disclosure provides a method of, or assay for,identifying a subject having cancer (e.g., a hematological cancer suchas CLL and ALL) as having an increased or decreased likelihood torespond to a treatment that comprises a CAR-expressing cell therapy(e.g., a CD19 CAR-expressing cell therapy described herein such as,e.g., CTL019), the method comprising:

-   -   (1) acquiring a sample from the subject;    -   (2) determining a level of one or more (e.g., 2, 3, 4, 5, 6, 7,        8, 9, 10, 15 or more) markers listed in Table 1A, Table 1B,        Table 7A, Table 7B, Table 8, Table 9, Table 10, Table 14, Table        15, Table 16 (e.g., CCL20, IL-17a and/or IL-6), Table 17, Table        18, Table 20, PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L, and        KLRG1 in the sample; and    -   (3) comparing the determined level of one or more markers to a        reference level; wherein a difference, e.g., statistically        significant difference in the determined level to the reference        level is predictive of the subjects responsiveness to the        CAR-expressing cell therapy; and    -   (4) identifying the subject as a complete responder, partial        responder or non-responder to the CAR-expressing cell therapy.

In one embodiment, the sample is a blood, plasma or a serum sample. Inone embodiment, the sample is an apheresis sample, e.g., T cellsobtained from the blood of the subject. In an embodiment, the sample isa manufactured product sample, e.g., genetically engineered T cellsobtained from the blood of the subject, e.g., a manufacturedCAR-expressing cell product, e.g., a manufactured CD19 CAR-expressingcell product.

In an embodiment, the disclosure provides a method of, or assay for,identifying a subject having a cancer including, but not limited to,B-cell acute lymphocytic leukemia (B-ALL), T-cell acute lymphocyticleukemia (T-ALL), acute lymphocytic leukemia (ALL), chronic myelogenousleukemia (CML), chronic lymphocytic leukemia (CLL), B cell promyelocyticleukemia, blastic plasmacytoid dendritic cell neoplasm, Burkittslymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cellleukemia, small cell- or a large cell-follicular lymphoma, malignantlymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma(MCL), marginal zone lymphoma, multiple myeloma, myelodysplasia andmyelodysplastic syndrome, non-Hodgkin lymphoma, Hodgkin lymphoma,plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, andWaldenstrom macroglobulinemia. In an embodiment, the cancer is ahematological cancer. In an embodiment, the cancer is ALL. In anotherembodiment, the cancer is CLL. In an embodiment, the cancer isassociated with CD19 expression.

In an embodiment, a CAR-expressing cell therapy comprises aCAR-expressing cell therapy described herein, e.g., CTL019.

In an embodiment, a CAR-expressing cell therapy consists of aCAR-expressing cell therapy described herein, e.g., CTL019.

In an embodiment, the disclosure provides a method of, or assay for,identifying a subject having cancer (e.g., a hematological cancer suchas CLL and ALL) as having an increased or decreased likelihood torespond to a treatment that comprises a CAR-expressing cell therapy(e.g., a CD19 CAR-expressing cell therapy described herein such as,e.g., CTL019), the method comprising:

-   -   (1) acquiring a sample from the subject;    -   (2) determining a gene signature of the sample; and    -   (3) comparing the determined gene signature to a reference gene        signature;        wherein a difference, e.g., statistically significant difference        in expression level of one or more of the markers in the        determined gene signature, e.g., as compared to a predetermined        value, is predictive of the subjects responsiveness to the        CAR-expressing cell therapy.

In an embodiment, the disclosure provides a method of, or assay for,determining the responsiveness of a subject having cancer (e.g., ahematological cancer such as CLL and ALL) to a treatment comprising acell expressing a CAR (e.g., a cell expressing a CD19 CAR, e.g., a CD19CAR-expressing cell (e.g., T cell, NK cell) described herein such as,e.g., CTL019), the method comprising:

determining a level of one or more markers listed in Table 1A, Table 1B,Table 7A, Table 7B, Table 8, Table 9, Table 10, Table 14, Table 15,Table 16 (e.g., CCL20, IL-17a and/or IL-6), Table 17, Table 18, Table20, PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L, and KLRG1 in a sampleobtained prior to treatment;

wherein a statistically significant difference in expression level ofone or more markers in the sample relative to a predetermined value isindicative of increased responsiveness to the CAR-expressing cell.

The methods provided herein are particularly useful for identifyingsubjects that are likely to respond to CAR-expressing cell (e.g., Tcell, NK cell) therapy (e.g., a CD19 CAR-expressing cell therapydescribed herein such as, e.g., CTL019) prior to initiation of suchtreatment (e.g., pre-therapy) or early in the therapeutic regimen. Insome embodiments, expression or activity of biomarkers is measured in asubject at least 2 weeks, at least 1 month, at least 3 months, at least6 months, or at least 1 year prior to initiation of therapy. In someembodiments, expression or activity of biomarkers is measured less than6 months prior to the initiation of therapy. Thus, in some embodiments,expression or activity of biomarkers is measured within 6 months, 5months, 4 months, 3 months, 2 months, 1 month, 2 weeks, 1 week, 6 days,5 days, 4 days, 3 days, 2 days, or 1 day prior to the initiation oftherapy. In other embodiments, the expression or activity of biomarkersis determined after initiation of therapy (e.g., 1 month, 2 months, 3months, 3.5 months, 4 months, 4.5 months, 5 months, 5.5 months, 6months).

In an embodiment, the invention provides a method of evaluating asubject having cancer (e.g., a hematological cancer such as CLL and ALL)comprising:

acquiring a value of responder status for the subject that comprises ameasure of one or more of the following:

one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,45, 50 or more) biomarker listed in Table 1A, Table 1B, Table 7A, Table7B, Table 8, Table 9, Table 10, Table 14, Table 15, Table 16 (e.g.,CCL20, IL-17a and/or IL-6), Table 17, Table 18, Table 20, a CD27biomarker, a CD45RO biomarker, a PD-1 biomarker, a LAG-3 biomarker, aTIM-3 biomarker, an IL2RA biomarker, an IL21 biomarker, a CD4 biomarker,a CD8 biomarker, a TH1+ helper T cell gene set signature, a TH2+ helperT cell gene set signature, a memory T cell (e.g., a CD8+ memory T cell,e.g., a naïve T cell (T_(N)), e.g. a memory stem cell (T_(SCM)), e.g. acentral memory T cell (T_(CM)), e.g. an effector memory T cell (T_(EM)))gene set signature, and a CD19 CAR-expressing cell gene set signature,thereby evaluating the subject.

In an embodiment, the disclosure provides a method of evaluating asubject having cancer (e.g., a hematological cancer such as CLL and ALL)comprising acquiring a value of responder status for the subject thatcomprises a measure of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 35, 40, 45, 50 or more) of the following: a biomarkerlisted in Table 1A, Table 1B, Table 7A, Table 7B, Table 8, Table 9,Table 10, Table 14, Table 15, Table 16 (e.g., CCL20, IL-17a and/orIL-6), Table 17, Table 18, Table 20, PD-1, LAG-3, TIM-3, CD57, CD27,CD122, CD62L, KLRG1, and a CD19 CAR-expressing cell gene set signature,thereby evaluating the subject.

In an embodiment, the disclosure provides a method of evaluating ormonitoring the effectiveness of a CAR-expressing cell therapy in asubject having cancer comprising:

acquiring a value of responder status for the subject that comprises ameasure of one or more of the following:

a biomarker listed in Table 1A, Table 1B, Table 7A, Table 7B, Table 8,Table 9, Table 10, Table 14, Table 15, Table 16 (e.g., CCL20, IL-17aand/or IL-6), Table 17, Table 18, Table 20, a CD27 biomarker, a CD45RObiomarker, a PD-1 biomarker, a LAG-3 biomarker, a TIM-3 biomarker, anIL2RA biomarker, an IL21 biomarker, a CD4 biomarker, a CD8 biomarker, aTH1+ helper T cell gene set signature, a TH2+ helper T cell gene setsignature, a memory T cell (e.g., a CD8+ memory T cell, e.g., a naïve Tcell (T_(N)), e.g. a memory stem cell (T_(SCM)), e.g. a central memory Tcell (T_(CM)), e.g. an effector memory T cell (T_(EM))) gene setsignature, and a CD19 CAR-expressing cell gene set signature,

thereby evaluating or monitoring the effectiveness of the CAR therapy inthe subject.

In an embodiment, the disclosure provides a method of evaluating ormonitoring the effectiveness of a CAR-expressing cell (e.g., T cell, NKcell) therapy (e.g., a CD19 CAR-expressing cell therapy described hereinsuch as, e.g., CTL019) in a subject having cancer (e.g., a hematologicalcancer such as CLL and ALL) comprising: acquiring a value of responderstatus for the subject that comprises a measure of one or more (e.g., 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more) of thefollowing: a biomarker listed in Table 1A, Table 1B, Table 7A, Table 7B,Table 8, Table 9, Table 10, Table 14, Table 15, Table 16 (e.g., CCL20,IL-17a and/or IL-6), Table 17, Table 18, Table 20, PD-1, LAG-3, TIM-3,CD57, CD27, CD122, CD62L, KLRG1, and a CD19 CAR-expressing cell (e.g., Tcell, NK cell) gene set signature, thereby evaluating or monitoring theeffectiveness of the CAR-expressing cell (e.g., T cell, NK cell) therapyin the subject.

In an embodiment, the value of responder status comprises a measure of acombination of a gene signature and a biomarker.

In an embodiment, the value of the responder status comprises a measureof a CD19 CAR-expressing cell gene set signature and a combination ofone or more of: a biomarker listed in Table 1A, Table 1B, Table 7A,Table 7B, Table 8, Table 9, Table 10, Table 14, Table 15, Table 16(e.g., CCL20, IL-17a and/or IL-6), Table 17, Table 18, Table 20, a CD27biomarker, a CD45RO biomarker, a PD-1 biomarker, a LAG-3 biomarker, aTIM-3 biomarker, an IL2RA biomarker, an IL21 biomarker, a CD4 biomarker,a CD8 biomarker, a TH1+ helper T cell gene set signature, a TH2+ helperT cell gene set signature, and a memory T cell (e.g., a CD8+ memory Tcell, e.g., a naïve T cell (T_(N)), e.g. a memory stem cell (T_(SCM)),e.g. a central memory T cell (T_(CM)), e.g. an effector memory T cell(T_(EM))) gene set signature.

In an embodiment, the value of the responder status comprises a measureof a CD19 CAR-expressing cell gene set signature and a combination ofone or more of: a biomarker listed in Table 1A, Table 1B, Table 7A,Table 7B, Table 8, Table 9, Table 10, Table 14, Table 15, Table 16(e.g., CCL20, IL-17a and/or IL-6), Table 17, Table 18, Table 20, PD-1,LAG-3, TIM-3, CD57, CD27, CD122, CD62L, and KLRG1.

In an embodiment, the value of the responder status comprises a measureof one or more biomarkers listed in Table 1A, Table 1B, Table 7A, Table7B and a combination of one or more of: a CD27 biomarker, a CD45RObiomarker, a PD-1 biomarker, a LAG-3 biomarker, a TIM-3 biomarker, anIL2RA biomarker, an IL21 biomarker, a CD4 biomarker, a CD8 biomarker, aTH1+ helper T cell gene set signature, a TH2+ helper T cell gene setsignature, a memory T cell (e.g., a CD8+ memory T cell, e.g., a naïve Tcell (T_(N)), e.g. a memory stem cell (T_(SCM)), e.g. a central memory Tcell (T_(CM)), e.g. an effector memory T cell (T_(EM))) gene setsignature, and a CD19 CAR-expressing cell gene set signature.

In an embodiment, the value of the responder status comprises a measureof one or more biomarkers listed in Table 1A, Table 1B, Table 7A, Table7B and a combination of one or more of: CD57, CD27, CD122, CD62L, andKLRG1.

In an embodiment, the CD19 CAR-expressing cell gene signature comprisesa value for expression of at least 5, 6, 7, 8, 9 or 10 genes comprisinga CD19 CAR-expressing cell gene signature.

In an embodiment, the value for expression of the gene comprises a valuefor a transcriptional parameter, e.g., the level of an mRNA encoded bythe gene.

In an embodiment, the value for expression of the protein comprises avalue for a translational parameter, e.g., the level of a protein.

In an embodiment, provided methods further comprise obtaining a samplefrom the subject, wherein the sample comprises a cellular or tissuefraction. In an embodiment, the cellular fraction comprises blood.

In an embodiment, the measure of biomarker and/or gene signature isacquired before, at the same time, or during course of a CAR-expressingcell therapy (e.g., a CD19 CAR-expressing cell therapy described hereinsuch as, e.g., CTL019).

In an embodiment, the measure of biomarker and/or gene signature isacquired less than 6 months, 5 months, 4 months, 3 months, 2 months, 1month, 2 weeks, 1 week, 6 days, 5 days, 4 days, 3 days, 2 days prior tothe initiation of a CAR-expressing cell therapy (e.g., a CD19CAR-expressing cell therapy described herein such as, e.g., CTL019).

The methods described herein can also be used to monitor a positiveresponse of a subject to CAR-expressing cell (e.g., T cell, NK cell)treatment (e.g., a CD19 CAR-expressing cell treatment described hereinsuch as, e.g., CTL019). Such methods are useful for early detection oftolerance to therapy or to predict whether disease in a subject willprogress. In such embodiments, the expression or activity of biomarkersis determined e.g., at least every week, at least every 2 weeks, atleast every month, at least every 2 months, at least every 3 months, atleast every 4 months, at least every 5 months, at least every 6 months,at least every 7 months, at least every 8 months, at least every 9months, at least every 10 months, at least every 11 months, at leastevery year, at least every 18 months, at least every 2 years, at leastevery 3 years, at least every 5 years or more. It is also contemplatedthat expression or activity of the biomarkers is at irregular intervalse.g., biomarkers can be detected in a subject at 3 months of treatment,at 6 months of treatment, and at 7 months of treatment. Thus, in someembodiments, the expression or activity of the biomarkers is determinedwhen deemed necessary by the skilled physician monitoring treatment ofthe subject.

The methods described herein can be used in treating any subject havingcancer (e.g., a hematological cancer such as CLL and ALL). In oneaspect, the invention pertains to methods of treating cancer (e.g., ahematological cancer such as CLL and ALL) in a subject.

In an embodiment, the disclosure provides methods for treating cancerincluding, but not limited to, B-cell acute lymphocytic leukemia(B-ALL), T-cell acute lymphocytic leukemia (T-ALL), acute lymphocyticleukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocyticleukemia (CLL), B cell promyelocytic leukemia, blastic plasmacytoiddendritic cell neoplasm, Burkitts lymphoma, diffuse large B celllymphoma, follicular lymphoma, hairy cell leukemia, small cell- or alarge cell-follicular lymphoma, malignant lymphoproliferativeconditions, MALT lymphoma, mantle cell lymphoma (MCL), marginal zonelymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome,non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma,plasmacytoid dendritic cell neoplasm, and Waldenstrom macroglobulinemia.In an embodiment, the invention provides methods for treating ALL. Inanother embodiment, the invention provides methods for treating CLL. Inan embodiment, the invention provides methods for treating cancer thatis associated with CD19 expression.

In an embodiment, provided methods comprise administering to the subjecta cell expressing a CAR, e.g. a CAR T cell, e.g. a CD19 CAR T cell,e.g., a CTL019 product, if the subject is identified as having adifference, e.g., statistically significant difference in expressionlevel of one or more markers listed in Table 1A, Table 1B, Table 7A,Table 7B, Table 8, Table 9, Table 10, Table 14, Table 15, Table 16(e.g., CCL20, IL-17a and/or IL-6), Table 17, Table 18, Table 20, PD-1,LAG-3, TIM-3, CD57, CD27, CD122, CD62L, and KLRG1 relative to areference level, such that the cancer (e.g., a hematological cancer suchas ALL and CLL) is treated in the subject.

As discussed above, an example of a cancer that is treatable bydisclosed methods is a cancer associated with expression of CD19. In oneaspect, the cancer associated with expression of CD19 is a hematologicalcancer. In one aspect, the hematological cancer is a leukemia or alymphoma. In one aspect, a cancer associated with expression of CD19includes cancers and malignancies including, but not limited to, e.g.,one or more acute leukemias including but not limited to, e.g., B-cellacute lymphoid leukemia (“B-ALL”), T-cell acute lymphoid leukemia(“T-ALL”), acute lymphoid leukemia (ALL); one or more chronic leukemiasincluding but not limited to, e.g., chronic myelogenous leukemia (CML),chronic lymphocytic leukemia (CLL). Additional cancers or hematologicconditions associated with expression of CD19 include, but are notlimited to, e.g., B cell promyelocytic leukemia, blastic plasmacytoiddendritic cell neoplasm, Burkitts lymphoma, diffuse large B celllymphoma, follicular lymphoma, hairy cell leukemia, small cell- or alarge cell-follicular lymphoma, malignant lymphoproliferativeconditions, MALT lymphoma, mantle cell lymphoma (MCL), marginal zonelymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome,non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma,plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and“preleukemia” which are a diverse collection of hematological conditionsunited by ineffective production (or dysplasia) of myeloid blood cells,and the like. Further, a disease associated with CD19 expressioninclude, but not limited to, e.g., atypical and/or non-classicalcancers, malignancies, precancerous conditions or proliferative diseasesassociated with expression of CD19.

In an embodiment, the disclosure provides a method for treating asubject having cancer (e.g., a hematological cancer such as ALL and CLL)comprising:

determining if the subject has an increased likelihood to respond to aCAR-expressing cell (e.g., T cell, NK cell) therapy (e.g., a CD19CAR-expressing cell therapy described herein such as, e.g., CTL019) bycomparing the level of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 35, 40, 45, 50 or more) markers in Table 1A, Table 1B,Table 7A, Table 7B, Table 8, Table 9, Table 10, Table 14, Table 15,Table 16 (e.g., CCL20, IL-17a and/or IL-6), Table 17, Table 18, Table20, PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L, and KLRG1 in a samplefrom the subject relative to a reference level, wherein a statisticallysignificant difference in expression level of one or more maker genesrelative to the reference level is indicative of an increased likelihoodof response; and

administering to the subject a therapeutically effective dose of aCAR-expressing cell therapy.

In an embodiment, the disclosure provides a method for treating asubject having cancer (e.g., a hematological cancer such as ALL andCLL), comprising:

obtaining a sample from the subject;

determining a level of one or more markers in Table 1A, Table 1B, Table7A, Table 7B, Table 8, Table 9, Table 10, Table 14, Table 15, Table 16(e.g., CCL20, IL-17a and/or IL-6), Table 17, Table 18, Table 20, PD-1,LAG-3, TIM-3, CD57, CD27, CD122, CD62L, and KLRG1 in the sample;

comparing the determined level of one or more markers in Table 1A, Table1B, Table 7A, Table 7B, Table 8, Table 9, Table 10, Table 14, Table 15,Table 16 (e.g., CCL20, IL-17a and/or IL-6), Table 17, Table 18, Table20, PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L, and KLRG1 to areference level; and

administering a therapeutically effective dose of a CAR-expressing cell(e.g., T cell, NK cell) therapy (e.g., a CD19 CAR-expressing celltherapy described herein such as, e.g., CTL019), if the subject isidentified as having a statistically significant difference in thedetermined level of one or more markers in Table 1A, Table 1B, Table 7A,Table 7B, Table 8, Table 9, Table 10, Table 14, Table 15, Table 16(e.g., CCL20, IL-17a and/or IL-6), Table 17, Table 18, Table 20, PD-1,LAG-3, TIM-3, CD57, CD27, CD122, CD62L, and KLRG1 to a reference level,in the sample.

In an embodiment, the disclosure provides a method of treating cancer(e.g., a hematological cancer such as ALL and CLL) in a subject,comprising:

acquiring a value of responder status for the subject that comprises ameasure of one or more of the following:

a biomarker listed in Table 1A, Table 1B, Table 7A, Table 7B, Table 8,Table 9, Table 10, Table 14, Table 15, Table 16 (e.g., CCL20, IL-17aand/or IL-6), Table 17, Table 18, Table 20, a CD27 biomarker, a CD45RObiomarker, a PD-1 biomarker, a LAG-3 biomarker, a TIM-3 biomarker, anIL2RA biomarker, an IL21 biomarker, a CD4 biomarker, a CD8 biomarker, aTH1+ helper T cell gene set signature, a TH2+ helper T cell gene setsignature, a memory T cell (e.g., a CD8+ memory T cell, e.g., a naïve Tcell (T_(N)), e.g. a memory stem cell (T_(SCM)), e.g. a central memory Tcell (T_(CM)), e.g. an effector memory T cell (T_(EM))) gene setsignature, and a CD19 CAR-expressing cell gene set signature, andresponsive to a determination of responder status, performing one, two,three, four or more of:

identifying the subject as a complete responder, partial responder ornon-responder;

administering a CAR-expressing cell therapy (e.g., a CD19 CAR-expressingcell therapy described herein such as, e.g., CTL019);

selecting or altering a dosing of a CAR-expressing cell therapy;

selecting or altering the schedule or time course of a CAR-expressingcell therapy;

administering, e.g., to a non-responder or a partial responder, anadditional agent in combination with a CAR-expressing cell therapy,e.g., a checkpoint inhibitor, e.g., a checkpoint inhibitor describedherein;

administering to a non-responder or partial responder a therapy thatincreases the number of naïve T cells in the subject prior to treatmentwith a CAR-expressing cell therapy;

modifying a manufacturing process of a CAR-expressing cell therapy,e.g., enrich for naïve T cells prior to introducing a nucleic acidencoding a CAR, e.g., for a subject identified as a non-responder or apartial responder; or selecting an alternative therapy, e.g., for anon-responder or partial responder; or

selecting an alternative therapy, e.g., an alternative therapy describedherein, e.g., a standard of care therapy for the cancer; therebytreating cancer in a subject.

In an embodiment, the disclosure provides a method of treating cancer(e.g., a hematological cancer such as ALL and CLL) in a subject,comprising: acquiring a value of responder status for the subject thatcomprises a measure of one or more of the following: a biomarker listedin Table 1A, Table 1B, Table 7A, Table 7B, Table 8, Table 9, Table 10,Table 14, Table 15, Table 16 (e.g., CCL20, IL-17a and/or IL-6), Table17, Table 18, Table 20, PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L,KLRG1, and a CD19 CAR-expressing cell gene set signature, and responsiveto a determination of responder status, performing one, two, three fouror more of: identifying the subject as a complete responder, partialresponder or non-responder; administering a CAR-expressing cell therapy(e.g., a CD19 CAR-expressing cell therapy described herein such as,e.g., CTL019); selecting or altering a dosing of a CAR-expressing celltherapy; selecting or altering the schedule or time course of aCAR-expressing cell therapy; administering, e.g., to a non-responder ora partial responder, an additional agent in combination with aCAR-expressing cell therapy, e.g., a checkpoint inhibitor, e.g., acheckpoint inhibitor described herein; administering to a non-responderor partial responder a therapy that increases the number of naïve Tcells in the subject prior to treatment with a CAR-expressing celltherapy; modifying a manufacturing process of a CAR-expressing celltherapy, e.g., enrich for naïve T cells prior to introducing a nucleicacid encoding a CAR into the T cells, e.g., for a subject identified asa non-responder or a partial responder; selecting an alternativetherapy, e.g., a standard of care for the cancer, e.g., for anon-responder or partial responder; or selecting an alternativeCAR-expressing cell therapy; thereby treating cancer in a subject.

In some embodiments, the amount of the biomarker determined in a samplefrom a subject is quantified as an absolute measurement (e.g., ng/mL).Absolute measurements can easily be compared to a reference value orcut-off value. For example, a cut-off value can be determined thatrepresents a disease progressing status; any absolute values fallingeither above (i.e., for biomarkers that increase expression withprogression of a cancer, e.g., a hematological cancer such as ALL andCLL) or falling below (i.e., for biomarkers with decreased expressionwith progression of a cancer, e.g., a hematological cancer such as ALLand CLL) the cut-off value are likely to be disease progressing.

Alternatively, the relative amount of a biomarker is determined. In oneembodiment, the relative amount is determined by comparing theexpression and/or activity of one or more biomarkers in a subject withcancer to the expression of the biomarkers in a reference parameter. Insome embodiments, a reference parameter is obtained from one or more of:a baseline or prior value for the subject, the subject at a differenttime interval, an average or median value for a cancer subject (e.g.,patient) population, a healthy control, or a healthy subject population.

The present disclosure also pertains to the field of predictive medicinein which diagnostic assays, pharmacogenomics, and monitoring clinicaltrials are used for predictive purposes to thereby treat an individualprophylactically. Accordingly, one aspect of the present disclosurerelates to assays for determining the amount, structure, and/or activityof polypeptides or nucleic acids corresponding to one or more markersdescribed herein, in order to determine whether an individual havingcancer (e.g., a hematological cancer such as CLL and ALL) or at risk ofdeveloping cancer (e.g., a hematological cancer such as CLL and ALL)will be more likely to respond to CAR-expressing cell therapy (e.g., aCD19 CAR-expressing cell therapy described herein such as, e.g.,CTL019).

Accordingly, in one aspect, the disclosure provides a method fordetermining whether a subject with cancer (e.g., a hematological cancersuch as CLL and ALL) is likely to respond to a cell expressing a CAR,e.g., a CD19 CAR-expressing cell described herein, such as CTL019. Inanother aspect, the disclosure is drawn to a method for predicting atime course of disease. In still another aspect, the method is drawn toa method for predicting a probability of a significant event in the timecourse of the disease (e.g., reoccurrence or remission). In certainembodiments, the method comprises detecting a combination of biomarkersassociated with responsiveness to treatment as described herein anddetermining whether the subject is likely to respond to treatment.

In an aspect, the disclosure provides a method for providing a prognosisfor success rate of a CAR-expressing cell therapy (e.g., a CD19CAR-expressing cell therapy described herein such as, e.g., CTL019) in asubject having cancer (e.g., a hematological cancer such as ALL andCLL), said method comprising steps of:

providing a biological sample from the subject;

determining the levels of expression of one or more (e.g., 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20 or more) genes listed in Table 1A, Table 1B,Table 7A, Table 7B, Table 8, Table 9, Table 10, Table 14, Table 15,Table 16 (e.g., CCL20, IL-17a and/or IL-6), Table 17, Table 18, Table20, PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L, and KLRG1 to obtain agene expression pattern for the sample; and based on the gene expressionpattern obtained, providing a prognosis to the subject.

In an embodiment, the step of determining the levels of expression ofthe set of genes further comprises detecting the expression of mRNAexpressed from said genes. In an embodiment, provided methods furthercomprise a step wherein determining the expression of mRNA comprisesexposing said mRNA to a nucleic acid probe complementary to said mRNA.

In an embodiment, the step of determining the levels of expression ofthe set of genes further comprises detecting the expression of apolypeptide encoded by said genes.

In an embodiment, provided methods comprise selecting a CAR-expressingcell (e.g., T cell, NK cell) therapy (e.g., a CD19 CAR-expressing celltherapy described herein such as, e.g., CTL019) for the subject, basedon the prognosis provided.

In some embodiments, the methods involve evaluation of a biologicalsample, e.g., a sample from a subject, e.g., a patient who has beendiagnosed with or is suspected of having cancer (e.g., a hematologicalcancer such as CLL or ALL, e.g., presents with symptoms of CLL or ALL)to detect changes in expression and/or activity of 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15 or more genes in Table 1A, Table 1B, Table 7A, Table 7B,Table 8, Table 9, Table 10, Table 14, Table 15, Table 16 (e.g., CCL20,IL-17a and/or IL-6), Table 17, Table 18, Table 20, PD-1, LAG-3, TIM-3,CD57, CD27, CD122, CD62L, and KLRG1 and a CD19 CAR-expressing cell(e.g., T cell, NK cell) gene signature.

The results of the screening method and the interpretation thereof arepredictive of the patient disease progression (e.g., progression of acancer, e.g., a hematological cancer such as ALL or CLL). According tothe present invention, alterations in expression or activity of 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 15 or more genes in Table 1A, Table 1B, Table 7A,Table 7B, Table 8, Table 9, Table 10, Table 14, Table 15, Table 16(e.g., CCL20, IL-17a and/or IL-6), Table 17, Table 18, Table 20, PD-1,LAG-3, TIM-3, CD57, CD27, CD122, CD62L, and KLRG1 and a CD19CAR-expressing cell (e.g., T cell, NK cell) gene signature is indicativeof cancer progression (e.g., a hematological cancer such as ALL and CLL)relative to an average or median value for a cancer patient populationor to an average median for a population of healthy, cancer freesubjects.

In yet another embodiment, the one or more alterations, e.g.,alterations in biomarker expression are assessed at pre-determinedintervals, e.g., a first point in time and at least at a subsequentpoint in time. In one embodiment, a time course is measured bydetermining the time between significant events in the course of asubject disease, wherein the measurement is predictive of whether asubject has a long time course. In another embodiment, the significantevent is the progression from diagnosis to death. In another embodiment,the significant event is the progression from diagnosis to worseningdisease.

Methods for Detection of Gene Expression

Biomarker expression level can also be assayed. Expression of a markerdescribed herein can be assessed by any of a wide variety of knownmethods for detecting expression of a transcribed molecule or protein.Non-limiting examples of such methods include immunological methods fordetection of secreted, cell-surface, cytoplasmic, or nuclear proteins,protein purification methods, protein function or activity assays,nucleic acid hybridization methods, nucleic acid reverse transcriptionmethods, and nucleic acid amplification methods.

In certain embodiments, activity of a particular gene is characterizedby a measure of gene transcript (e.g., mRNA), by a measure of thequantity of translated protein, or by a measure of gene productactivity. Marker expression can be monitored in a variety of ways,including by detecting mRNA levels, protein levels, or protein activity,any of which can be measured using standard techniques. Detection caninvolve quantification of the level of gene expression (e.g., genomicDNA, CDNA, mRNA, protein, or enzyme activity), or, alternatively, can bea qualitative assessment of the level of gene expression, in particularin comparison with a control level. The type of level being detectedwill be clear from the context.

Methods of detecting and/or quantifying the gene transcript (mRNA orCDNA made therefrom) using nucleic acid hybridization techniques areknown to those of skill in the art (see e.g., Sambrook et al. supra).For example, one method for evaluating the presence, absence, orquantity of CDNA involves a Southern transfer as described above.Briefly, the mRNA is isolated (e.g., using an acidguanidinium-phenol-chloroform extraction method, Sambrook et al. supra.)and reverse transcribed to produce CDNA. The CDNA is then optionallydigested and run on a gel in buffer and transferred to membranes.Hybridization is then carried out using the nucleic acid probes specificfor the target CDNA.

A general principle of such diagnostic and prognostic assays involvespreparing a sample or reaction mixture that can contain a marker, and aprobe, under appropriate conditions and for a time sufficient to allowthe marker and probe to interact and bind, thus forming a complex thatcan be removed and/or detected in the reaction mixture. These assays canbe conducted in a variety of ways.

For example, one method to conduct such an assay would involve anchoringthe marker or probe onto a solid phase support, also referred to as asubstrate, and detecting target marker/probe complexes anchored on thesolid phase at the end of the reaction. In one embodiment of such amethod, a sample from a subject, which is to be assayed for presenceand/or concentration of marker, can be anchored onto a carrier or solidphase support. In another embodiment, the reverse situation is possible,in which the probe can be anchored to a solid phase and a sample from asubject can be allowed to react as an unanchored component of the assay.

In order to conduct assays with the above-mentioned approaches, thenon-immobilized component is added to the solid phase upon which thesecond component is anchored. After the reaction is complete,uncomplexed components can be removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized uponthe solid phase. The detection of marker/probe complexes anchored to thesolid phase can be accomplished in a number of methods outlined herein.

In another embodiment, the probe, when it is the unanchored assaycomponent, can be labeled for the purpose of detection and readout ofthe assay, either directly or indirectly, with detectable labelsdiscussed herein and which are well-known to one skilled in the art.

It is also possible to directly detect marker/probe complex formationwithout further manipulation or labeling of either component (marker orprobe), for example by utilizing the technique of fluorescence energytransfer (see, for example, Lakowicz et al., U.S. Pat. No. 5,631,169;Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). A fluorophore labelon the first, ‘donor’ molecule is selected such that, upon excitationwith incident light of appropriate wavelength, its emitted fluorescentenergy will be absorbed by a fluorescent label on a second ‘acceptor’molecule, which in turn is able to fluoresce due to the absorbed energy.Alternately, the ‘donor’ protein molecule can simply utilize the naturalfluorescent energy of tryptophan residues. Labels are chosen that emitdifferent wavelengths of light, such that the ‘acceptor’ molecule labelcan be differentiated from that of the ‘donor’. Since the efficiency ofenergy transfer between the labels is related to the distance separatingthe molecules, spatial relationships between the molecules can beassessed. In a situation in which binding occurs between the molecules,the fluorescent emission of the ‘acceptor’ molecule label in the assayshould be maximal. An FET binding event can be conveniently measuredthrough standard fluorometric detection means well known in the art(e.g., using a fluorimeter).

In another embodiment, determination of the ability of a probe torecognize a marker can be accomplished without labeling either assaycomponent (probe or marker) by utilizing a technology such as real-timeBiomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. andUrbaniczky, C., 1991, ANAL. CHEM. 63:2338-2345 and Szabo et al., 1995,CURR. OPIN. STRUCT. BIOL. 5:699-705). As used herein, “BIA” or “surfaceplasmon resonance” is a technology for studying biospecific interactionsin real time, without labeling any of the interactants (e.g., BIAcore).Changes in the mass at the binding surface (indicative of a bindingevent) result in alterations of the refractive index of light near thesurface (the optical phenomenon of surface plasmon resonance (SPR)),resulting in a detectable signal which can be used as an indication ofreal-time reactions between biological molecules.

Alternatively, in another embodiment, analogous diagnostic andprognostic assays can be conducted with marker and probe as solutes in aliquid phase. In such an assay, the complexed marker and probe areseparated from uncomplexed components by any of a number of standardtechniques, including but not limited to: differential centrifugation,chromatography, electrophoresis and immunoprecipitation. In differentialcentrifugation, marker/probe complexes can be separated from uncomplexedassay components through a series of centrifugal steps, due to thedifferent sedimentation equilibria of complexes based on their differentsizes and densities (see, for example, Rivas, G., and Minton, A. P.,1993, Trends Biochem Sci. 18(8):284-7). Standard chromatographictechniques can also be utilized to separate complexed molecules fromuncomplexed ones. For example, gel filtration chromatography separatesmolecules based on size, and through the utilization of an appropriategel filtration resin in a column format, for example, the relativelylarger complex can be separated from the relatively smaller uncomplexedcomponents. Similarly, the relatively different charge properties of themarker/probe complex as compared to the uncomplexed components can beexploited to differentiate the complex from uncomplexed components, forexample, through the utilization of ion-exchange chromatography resins.Such resins and chromatographic techniques are well known to one skilledin the art (see, e.g., Heegaard, N. H., 1998, J. MOL. RECOGNIT. Winter11(1-6):141-8; Hage, D. S., and Tweed, S. A. J CHROMATOGR B BIOMED SCIAPPL 1997 Oct. 10; 699(1-2):499-525). Gel electrophoresis can also beemployed to separate complexed assay components from unbound components(see, e.g., Ausubel et al., ed., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,John Wiley & Sons, New York, 1987-1999). In this technique, protein ornucleic acid complexes are separated based on size or charge, forexample. In order to maintain the binding interaction during theelectrophoretic process, non-denaturing gel matrix materials andconditions in the absence of reducing agent are typical. Appropriateconditions to the particular assay and components thereof will be wellknown to one skilled in the art.

In a particular embodiment, the level of mRNA corresponding to themarker can be determined both by in situ and by in vitro formats in abiological sample using methods known in the art. The term “biologicalsample” is intended to include tissues, cells, biological fluids andisolates thereof, isolated from a subject, as well as tissues, cells andfluids present within a subject. Many expression detection methods useisolated RNA. For in vitro methods, any RNA isolation technique thatdoes not select against the isolation of mRNA can be utilized for thepurification of RNA from cells (see, e.g., Ausubel et al., ed., CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York 1987-1999).Additionally, large numbers of tissue samples can readily be processedusing techniques well known to those of skill in the art, such as, forexample, the single-step RNA isolation process of Chomczynski (1989,U.S. Pat. No. 4,843,155).

The isolated nucleic acid can be used in hybridization or amplificationassays that include, but are not limited to, Southern or Northernanalyses, polymerase chain reaction analyses and probe arrays. Onediagnostic method for the detection of mRNA levels involves contactingthe isolated mRNA with a nucleic acid molecule (probe) that canhybridize to the mRNA encoded by the gene being detected. The nucleicacid probe can be, for example, a full-length CDNA, or a portionthereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250or 500 nucleotides in length and sufficient to specifically hybridizeunder stringent conditions to a mRNA or genomic DNA encoding a marker ofthe present invention. Other suitable probes for use in the diagnosticassays are described herein. Hybridization of an mRNA with the probeindicates that the marker in question is being expressed.

In one format, the mRNA is immobilized on a solid surface and contactedwith a probe, for example by running the isolated mRNA on an agarose geland transferring the mRNA from the gel to a membrane, such asnitrocellulose. In an alternative format, the probe(s) are immobilizedon a solid surface and the mRNA is contacted with the probe(s), forexample, in an Affymetrix gene chip array. A skilled artisan can readilyadapt known mRNA detection methods for use in detecting the level ofmRNA encoded by the markers described herein.

The probes can be full length or less than the full length of thenucleic acid sequence encoding the protein. Shorter probes areempirically tested for specificity. Exemplary nucleic acid probes are 20bases or longer in length (See, e.g., Sambrook et al. for methods ofselecting nucleic acid probe sequences for use in nucleic acidhybridization). Visualization of the hybridized portions allows thequalitative determination of the presence or absence of CDNA.

An alternative method for determining the level of a transcriptcorresponding to a marker of the present invention in a sample involvesthe process of nucleic acid amplification, e.g., by rtPCR (theexperimental embodiment set forth in Mullis, 1987, U.S. Pat. No.4,683,202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad. Sci.USA, 88:189-193), self-sustained sequence replication (Guatelli et al.,1990, PROC. NATL. ACAD. SCI. USA 87:1874-1878), transcriptionalamplification system (Kwoh et al., 1989, PROC. NATL. ACAD. SCI. USA86:1173-1177), Q-Beta Replicase (Lizardi et al., 1988, BIO/TECHNOLOGY6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No.5,854,033) or any other nucleic acid amplification method, followed bythe detection of the amplified molecules using techniques well known tothose of skill in the art. Fluorogenic rtPCR can also be used in themethods of the invention. In fluorogenic rtPCR, quantitation is based onamount of fluorescence signals, e.g., TaqMan and sybr green. Thesedetection schemes are especially useful for the detection of nucleicacid molecules if such molecules are present in very low numbers. Asused herein, amplification primers are defined as being a pair ofnucleic acid molecules that can anneal to 5′ or 3′ regions of a gene(plus and minus strands, respectively, or vice-versa) and contain ashort region in between. In general, amplification primers are fromabout 10 to 30 nucleotides in length and flank a region from about 50 to200 nucleotides in length. Under appropriate conditions and withappropriate reagents, such primers permit the amplification of a nucleicacid molecule comprising the nucleotide sequence flanked by the primers.

For in situ methods, mRNA does not need to be isolated from the cellsprior to detection. In such methods, a cell or tissue sample isprepared/processed using known histological methods. The sample is thenimmobilized on a support, typically a glass slide, and then contactedwith a probe that can hybridize to mRNA that encodes the marker.

As an alternative to making determinations based on the absoluteexpression level of the marker, determinations can be based on thenormalized expression level of the marker. Expression levels arenormalized by correcting the absolute expression level of a marker bycomparing its expression to the expression of a gene that is not amarker, e.g., a housekeeping gene that is constitutively expressed.Suitable genes for normalization include housekeeping genes such as theactin gene, or epithelial cell-specific genes. This normalization allowsthe comparison of the expression level in one sample, e.g., a subjectsample, to another sample, e.g., a healthy subject, or between samplesfrom different sources.

Alternatively, the expression level can be provided as a relativeexpression level. To determine a relative expression level of a marker,the level of expression of the marker can be determined for 10 or moresamples of normal versus cancer isolates, or even 50 or more samples,prior to the determination of the expression level for the sample inquestion. The mean expression level of each of the genes assayed in thelarger number of samples can be determined and this can be used as abaseline expression level for the marker. The expression level of themarker determined for the test sample (absolute level of expression)then can be divided by the mean expression value obtained for thatmarker. This provides a relative expression level.

In certain embodiments, the samples used in the baseline determinationwill be from samples derived from a subject having cancer (e.g., ahematological cancer such as ALL and CLL) versus samples from a healthysubject of the same tissue type. The choice of the cell source isdependent on the use of the relative expression level. Using expressionfound in normal tissues as a mean expression score aids in validatingwhether the marker assayed is specific to the tissue from which the cellwas derived (versus normal cells). In addition, as more data isaccumulated, the mean expression value can be revised, providingimproved relative expression values based on accumulated data.Expression data from normal cells provides a means for grading theseverity of the cancer disease state.

In another embodiment, expression of a marker is assessed by preparinggenomic DNA or mRNA/CDNA (i.e., a transcribed polynucleotide) from cellsin a subject sample, and by hybridizing the genomic DNA or mRNA/CDNAwith a reference polynucleotide which is a complement of apolynucleotide comprising the marker, and fragments thereof. CDNA can,optionally, be amplified using any of a variety of polymerase chainreaction methods prior to hybridization with the referencepolynucleotide. Expression of one or more markers can likewise bedetected using quantitative PCR (QPCR) to assess the level of expressionof the marker(s). Alternatively, any of the many known methods ofdetecting mutations or variants (e.g., single nucleotide polymorphisms,deletions, etc.) of a marker of the invention can be used to detectoccurrence of a mutated marker in a subject.

In a related embodiment, a mixture of transcribed polynucleotidesobtained from the sample is contacted with a substrate having fixedthereto a polynucleotide complementary to or homologous with at least aportion (e.g., at least 7, at least 10, at least 15, at least 20, atleast 25, at least 30, at least 40, at least 50, at least 100, at least500, or more nucleotide residues) of a marker described herein. Ifpolynucleotides complementary to, or homologous with, a marker describedherein are differentially detectable on the substrate (e.g., detectableusing different chromophores or fluorophores, or fixed to differentselected positions), then the levels of expression of a plurality ofmarkers can be assessed simultaneously using a single substrate (e.g., a“gene chip” microarray of polynucleotides fixed at selected positions).When a method of assessing marker expression is used which involveshybridization of one nucleic acid with another, the hybridization can beperformed under stringent hybridization conditions.

In another embodiment, a combination of methods to assess the expressionof a marker is utilized.

Because the compositions, kits, and methods rely on detection of adifference in expression levels of one or more markers described herein,in certain embodiments the level of expression of the marker issignificantly greater than the minimum detection limit of the methodused to assess expression in at least one of a biological sample from asubject with cancer (e.g., a hematological cancer such as ALL and CLL)or a reference (e.g., a biological sample from a healthy subject, e.g.,a subject without cancer).

Nucleic Acid Molecules and Probes

One aspect of the disclosure pertains to isolated nucleic acid moleculesthat correspond to one or markers described herein, including nucleicacids which encode a polypeptide corresponding to one or more markersdescribed herein or a portion of such a polypeptide. The nucleic acidmolecules include those nucleic acid molecules which reside in genomicregions identified herein. Isolated nucleic acid molecules also includenucleic acid molecules sufficient for use as hybridization probes toidentify nucleic acid molecules that correspond to a marker describedherein, including nucleic acid molecules which encode a polypeptidecorresponding to a marker described herein, and fragments of suchnucleic acid molecules, e.g., those suitable for use as PCR primers forthe amplification or mutation of nucleic acid molecules. Nucleic acidmolecules can be DNA molecules (e.g., CDNA or genomic DNA) and RNAmolecules (e.g., mRNA) and analogs of the DNA or RNA generated usingnucleotide analogs. The nucleic acid molecule can be single-stranded ordouble-stranded; in certain embodiments the nucleic acid molecule isdouble-stranded DNA.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid molecule. In certain embodiments, an “isolated” nucleicacid molecule is free of sequences (such as protein-encoding sequences)which naturally flank the nucleic acid (i.e., sequences located at the 5and 3 ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived.

The language “substantially free of other cellular material or culturemedium” includes preparations of nucleic acid molecule in which themolecule is separated from cellular components of the cells from whichit is isolated or recombinantly produced. Thus, nucleic acid moleculethat is substantially free of cellular material includes preparations ofnucleic acid molecule having less than about 30%, less than about 20%,less than about 10%, or less than about 5% (by dry weight) of othercellular material or culture medium.

If so desired, a nucleic acid molecule, e.g., the marker gene productsidentified herein, can be isolated using standard molecular biologytechniques and the sequence information in the database recordsdescribed herein. Using all or a portion of such nucleic acid sequences,nucleic acid molecules can be isolated using standard hybridization andcloning techniques (e.g., as described in Sambrook et al., ed.,MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989).

A nucleic acid molecule can be amplified using CDNA, mRNA, or genomicDNA as a template and appropriate oligonucleotide primers according tostandard PCR amplification techniques. The nucleic acid molecules soamplified can be cloned into an appropriate vector and characterized byDNA sequence analysis. Furthermore, oligonucleotides corresponding toall or a portion of a nucleic acid molecule of the invention can beprepared by standard synthetic techniques, e.g., using an automated DNAsynthesizer.

Probes based on the sequence of a nucleic acid molecule of the inventioncan be used to detect transcripts (e.g., mRNA) or genomic sequencescorresponding to one or more markers described herein. The probecomprises a label group attached thereto, e.g., a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as part of a diagnostic test kit for identifying cells ortissues which mis-express the protein, such as by measuring levels of anucleic acid molecule encoding the protein in a sample of cells from asubject, e.g., detecting mRNA levels or determining whether a geneencoding the protein has been mutated or deleted.

Polypeptide Detection

Methods to measure biomarkers described herein, include, but are notlimited to: Western blot, immunoblot, enzyme-linked immunosorbant assay(ELISA), radioimmunoassay (RIA), immunoprecipitation, surface plasmonresonance, chemiluminescence, fluorescent polarization, phosphorescence,immunohistochemical analysis, liquid chromatography mass spectrometry(LC-MS), matrix-assisted laser desorption/ionization time-of-flight(MALDI-TOF) mass spectrometry, microcytometry, microarray, microscopy,fluorescence activated cell sorting (FACS), magnetic activated cellsorting (MACS), flow cytometry, laser scanning cytometry, hematologyanalyzer and assays based on a property of the protein including but notlimited to DNA binding, ligand binding, or interaction with otherprotein partners.

The activity or level of a marker protein can also be detected and/orquantified by detecting or quantifying the expressed polypeptide. Thepolypeptide can be detected and quantified by any of a number of meanswell known to those of skill in the art. These can include analyticbiochemical methods such as electrophoresis, capillary electrophoresis,high performance liquid chromatography (HPLC), thin layer chromatography(TLC), hyperdiffusion chromatography, and the like, or variousimmunological methods such as fluid or gel precipitin reactions,immunodiffusion (single or double), immunoelectrophoresis,radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, Western blotting, immunohistochemistry and thelike. A skilled artisan can readily adapt known protein/antibodydetection methods for use in determining the expression level of one ormore biomarkers in a serum sample.

Another agent for detecting a polypeptide is an antibody capable ofbinding to a polypeptide corresponding to a marker described herein,e.g., an antibody with a detectable label. Antibodies can be polyclonalor monoclonal. An intact antibody, or a fragment thereof (e.g., Fab orF(ab)₂) can be used. The term “labeled”, with regard to the probe orantibody, is intended to encompass direct labeling of the probe orantibody by coupling (i.e., physically linking) a detectable substanceto the probe or antibody, as well as indirect labeling of the probe orantibody by reactivity with another reagent that is directly labeled.Examples of indirect labeling include detection of a primary antibodyusing a fluorescently labeled secondary antibody and end-labeling of aDNA probe with biotin such that it can be detected with fluorescentlylabeled streptavidin.

In another embodiment, the antibody is labeled, e.g., a radio-labeled,chromophore-labeled, fluorophore-labeled, or enzyme-labeled antibody. Inanother embodiment, an antibody derivative (e.g., an antibody conjugatedwith a substrate or with the protein or ligand of a protein-ligand pair(e.g., biotin-streptavidin)), or an antibody fragment (e.g., asingle-chain antibody, an isolated antibody hypervariable domain, etc.)which binds specifically with a protein corresponding to the marker,such as the protein encoded by the open reading frame corresponding tothe marker or such a protein which has undergone all or a portion of itsnormal post-translational modification, is used.

Proteins from cells can be isolated using techniques that are well knownto those of skill in the art. The protein isolation methods employedcan, for example, be such as those described in Harlow and Lane (Harlowand Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.).

In one format, antibodies, or antibody fragments, can be used in methodssuch as Western blots or immunofluorescence techniques to detect theexpressed proteins. In such uses, one can immobilize either the antibodyor proteins on a solid support. Suitable solid phase supports orcarriers include any support capable of binding an antigen or anantibody. Well-known supports or carriers include glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, gabbros, and magnetite.

In another embodiment, the polypeptide is detected using an immunoassay.As used herein, an immunoassay is an assay that utilizes an antibody tospecifically bind to the analyte. The immunoassay is thus characterizedby detection of specific binding of a polypeptide to an anti-antibody asopposed to the use of other physical or chemical properties to isolate,target, and quantify the analyte.

The polypeptide is detected and/or quantified using any of a number ofwell recognized immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of thegeneral immunoassays, see also Asai (1993) Methods in Cell BiologyVolume 37: Antibodies in Cell Biology, Academic Press, Inc. New York;Stites & Terr (1991) Basic and Clinical Immunology 7th Edition.

In another embodiment, the polypeptide is detected and/or quantifiedusing Luminex® assay technology. The Luminex® assay separates tinycolor-coded beads into e.g., distinct sets that are each coated with areagent for a particular bioassay, allowing the capture and detection ofspecific analytes from a sample in a multiplex manner. The Luminex®assay technology can be compared to a multiplex ELISA assay usingbead-based fluorescence cytometry to detect analytes such as biomarkers.

The disclosure also encompasses kits for detecting the presence of apolypeptide or nucleic acid corresponding to a marker described hereinin a biological sample, e.g., a sample containing tissue, whole blood,serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool,and bone marrow. Such kits can be used to determine if a subject issuffering from or is at increased risk of developing cancer (e.g., ahematological cancer such as CLL and ALL). For example, the kit cancomprise a labeled compound or agent capable of detecting a polypeptideor an mRNA encoding a polypeptide corresponding to a marker describedherein in a biological sample and means for determining the amount ofthe polypeptide or mRNA in the sample (e.g., an antibody which binds thepolypeptide or an oligonucleotide probe which binds to DNA or mRNAencoding the polypeptide). Kits can also include instructions forinterpreting the results obtained using the kit.

The disclosure thus includes a kit for assessing the disease progressionof a subject having cancer (e.g., a hematological cancer such as CLL andALL).

In an embodiment, a kit can be used to assess the disease progression ofa cancer including, but not limited to, B-cell acute lymphocyticleukemia (B-ALL), T-cell acute lymphocytic leukemia (T-ALL), acutelymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), chroniclymphocytic leukemia (CLL), B cell promyelocytic leukemia, blasticplasmacytoid dendritic cell neoplasm, Burkitts lymphoma, diffuse large Bcell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- ora large cell-follicular lymphoma, malignant lymphoproliferativeconditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma,multiple myeloma, myelodysplasia and myelodysplastic syndrome,non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma,plasmacytoid dendritic cell neoplasm, and Waldenstrom macroglobulinemia.In an embodiment, the disclosure provides a kit for assessing thedisease progression of a subject having a hematological cancer. In anembodiment, the disclosure provides a kit for assessing the diseaseprogression of a subject having ALL. In another embodiment, thedisclosure provides a kit for assessing the disease progression of asubject having CLL. In an embodiment, the disclosure provides a kit forassessing the disease progression of a subject having cancer that isassociated with CD19 expression.

In an embodiment, the disclosure provides a kit for assessing andcharacterizing responder status (e.g., compete responder, partialresponder or non-responder) of a subject having a hematological cancerto a CAR-expressing cell (e.g., T cell, NK cell) therapy (e.g., a CD19CAR-expressing cell therapy as described herein, such as e.g., CTL019).In an embodiment, the disclosure provides a kit for assessing andcharacterizing responder status (e.g., compete responder, partialresponder or non-responder) of a subject having ALL to a CAR-expressingcell therapy (e.g., a CD19 CAR-expressing cell therapy as describedherein, such as e.g., CTL019). In an embodiment, the disclosure providesa kit for assessing and characterizing responder status (e.g., competeresponder, partial responder or non-responder) of a subject having CLLto a CAR-expressing cell therapy (e.g., a CD19 CAR-expressing celltherapy as described herein, such as e.g., CTL019).

Suitable reagents for binding with a polypeptide corresponding to amarker described herein include antibodies, antibody derivatives,antibody fragments, and the like. Suitable reagents for binding with anucleic acid (e.g., a genomic DNA, an mRNA, a spliced mRNA, a CDNA, orthe like) include complementary nucleic acids. For example, the nucleicacid reagents can include oligonucleotides (labeled or non-labeled)fixed to a substrate, labeled oligonucleotides not bound with asubstrate, pairs of PCR primers, molecular beacon probes, and the like.

The kit can optionally comprise additional components useful forperforming the methods described herein. By way of example, the kit cancomprise fluids (e.g., SSC buffer) suitable for annealing complementarynucleic acids or for binding an antibody with a protein with which itspecifically binds, one or more sample compartments, an instructionalmaterial which describes performance of a method of the invention, areference sample for comparison of expression levels of the biomarkersdescribed herein, and the like.

A kit of the invention can comprise a reagent useful for determiningprotein level or protein activity of a marker.

In an embodiment, a kit is provided for providing a prognosis forsuccess rate of a CAR-expressing cell (e.g., T cell, NK cell) therapy(e.g., a CD19 CAR-expressing cell therapy as described herein, such ase.g., CTL019) in a subject having cancer (e.g., a hematological cancersuch as CLL and ALL), said kit comprising:

a set of reagents that specifically detects expression levels of one ormore (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50 ormore) genes listed in Table 1A, Table 1B, Table 7A, Table 7B, Table 8,Table 9, Table 10, Table 14, Table 15, Table 16 (e.g., CCL20, IL-17aand/or IL-6), Table 17, Table 18, Table 20, PD-1, LAG-3, TIM-3, CD57,CD27, CD122, CD62L, and KLRG1 and a CD19 CAR-expressing cell gene setsignature; and

instructions for using said kit;

wherein said instructions for use provide that if one or more of thedetected expression levels is greater than a reference level, thesubject is more likely to respond positively to a CAR-expressing celltherapy.

In an embodiment, the set of reagents detects the expression of mRNAexpressed from said set of genes.

In an embodiment, the set of reagents comprises nucleic acid probescomplementary to mRNA expressed from said set of genes.

In an embodiment, the nucleic acid probes complementary to mRNA are CDNAor oligonucleotides.

In an embodiment, the nucleic acid probes complementary to mRNA areimmobilized on a substrate surface.

In an embodiment, the set of reagents detects the expression ofpolypeptides encoded by said set of genes.

Therapeutic Agents, Compositions and Administration

The methods described herein can be used to assess a responder status toa cell expressing a CAR. In one embodiment, the cell expresses a CARmolecule comprising an antigen binding domain (e.g., an antibody orantibody fragment that specifically binds to a tumor antigen), atransmembrane domain, and an intracellular signaling domain (e.g., anintracellular signaling domain comprising a costimulatory domain and/ora primary signaling domain). In an embodiment, the antigen bindingdomain comprises any antibody, or a fragment thereof, e.g., an scFv,known in the art that targets or specifically binds to any of the tumorantigens described herein. For example, the tumor antigen is BCMA (alsoknown as TNFRSF17, Tumor Necrosis Factor Receptor Superfamily, Member17, or B Cell Maturation Antigen), CD33, CLL-1 (also known as C-typeLectin-Like domain family 1, or CLECL1) or claudin-6 (CLDN6). Theantibody, or fragment thereof, can be a murine, humanized, or fullyhuman antibody or fragment thereof, e.g., an scFv.

In one embodiment, the CAR comprises an antibody or antibody fragmentwhich includes an anti-CD19 binding domain described herein (e.g., amurine or humanized antibody or antibody fragment that specificallybinds to CD19 as described herein), a transmembrane domain describedherein, and an intracellular signaling domain described herein (e.g., anintracellular signaling domain comprising a costimulatory domain and/ora primary signaling domain described herein).

Antigen Binding Domain

In one aspect, the CAR of the invention comprises a target-specificbinding element otherwise referred to as an antigen binding domain. Thechoice of moiety depends upon the type and number of ligands that definethe surface of a target cell. For example, the antigen binding domainmay be chosen to recognize a ligand that acts as a cell surface markeron target cells associated with a particular disease state. Thus,examples of cell surface markers that may act as ligands for the antigenbinding domain in a CAR of the invention include those associated withviral, bacterial and parasitic infections, autoimmune disease and cancercells.

In one aspect, the CAR-mediated T-cell response can be directed to anantigen of interest by way of engineering an antigen binding domain thatspecifically binds a desired antigen into the CAR.

In one aspect, the portion of the CAR comprising the antigen bindingdomain comprises an antigen binding domain that targets a tumor antigen,e.g., a tumor antigen described herein.

The antigen binding domain can be any domain that binds to the antigenincluding but not limited to a monoclonal antibody, a polyclonalantibody, a recombinant antibody, a human antibody, a humanizedantibody, and a functional fragment thereof, including but not limitedto a single-domain antibody such as a heavy chain variable domain (VH),a light chain variable domain (VL) and a variable domain (VHH) ofcamelid derived nanobody, and to an alternative scaffold known in theart to function as antigen binding domain, such as a recombinantfibronectin domain, a T cell receptor (TCR), or a fragment there of,e.g., single chain TCR, and the like. In some instances, it isbeneficial for the antigen binding domain to be derived from the samespecies in which the CAR will ultimately be used in. For example, foruse in humans, it may be beneficial for the antigen binding domain ofthe CAR to comprise human or humanized residues for the antigen bindingdomain of an antibody or antibody fragment.

Any known CD19 CAR, e.g., the CD19 antigen binding domain of any knownCD19 CAR, in the art can be used in accordance with the instantinvention. For example, LG-740; CD19 CAR described in the U.S. Pat. Nos.8,399,645; 7,446,190; Xu et al., Leuk Lymphoma. 201354(2):255-260(2012); Cruz et al., Blood 122(17):2965-2973 (2013);Brentjens et al., Blood, 118(18):4817-4828 (2011); Kochenderfer et al.,Blood 116(20):4099-102 (2010); Kochenderfer et al., Blood 122(25):4129-39(2013); and 16th Annu Meet Am Soc Gen Cell Ther (ASGCT) (May15-18, Salt Lake City) 2013, Abst 10.

Exemplary target antigens that can be targeted using the CAR-expressingcells, include, but are not limited to, CD19, CD123, EGFRvIII,mesothelin, among others, as described in, for example, WO 2014/130635,WO 2014/130657, and WO 2015/090230, each of which is herein incorporatedby reference in its entirety.

In one embodiment, the CAR T cell that specifically binds to CD19 hasthe USAN designation TISAGENLECLEUCEL-T. CTL019 is made by a genemodification of T cells is mediated by stable insertion via transductionwith a self-inactivating, replication deficient Lentiviral (LV) vectorcontaining the CTL019 transgene under the control of the EF-1 alphapromoter. CTL019 can be a mixture of transgene positive and negative Tcells that are delivered to the subject on the basis of percenttransgene positive T cells.

In other embodiments, the CAR-expressing cells can specifically bind tohuman CD19, e.g., can include a CAR molecule, or an antigen bindingdomain (e.g., a humanized antigen binding domain) according to Table 3of WO2014/153270, incorporated herein by reference.

In other embodiments, the CAR-expressing cells can specifically bind toCD123, e.g., can include a CAR molecule (e.g., any of the CAR1-CAR8), oran antigen binding domain according to Tables 1-2 of WO 2014/130635,incorporated herein by reference.

In other embodiments, the CAR-expressing cells can specifically bind toEGFRvIII, e.g., can include a CAR molecule, or an antigen binding domainaccording to Table 2 or SEQ ID NO:11 of WO 2014/130657, incorporatedherein by reference.

In other embodiments, the CAR-expressing cells can specifically bind tomesothelin, e.g., can include a CAR molecule, or an antigen bindingdomain according to Tables 2-3 of WO 2015/090230, incorporated herein byreference.

In one embodiment, the antigen binding domain comprises one, two three(e.g., all three) heavy chain CDRs, HC CDR1, HC CDR2 and HC CDR3, froman antibody listed above, and/or one, two, three (e.g., all three) lightchain CDRs, LC CDR1, LC CDR2 and LC CDR3, from an antibody listed above.In one embodiment, the antigen binding domain comprises a heavy chainvariable region and/or a variable light chain region of an antibodylisted or described above.

In some embodiments, the tumor antigen is a tumor antigen described inInternational Application WO2015/142675, filed Mar. 13, 2015, which isherein incorporated by reference in its entirety. In some embodiments,the tumor antigen is chosen from one or more of: CD19; CD123; CD22;CD30; CD171; CS-1 (also referred to as CD2 subset 1, CRACC, SLAMF7,CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1);CD33; epidermal growth factor receptor variant III (EGFRvIII);ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TNF receptor familymember B cell maturation (BCMA); Tn antigen ((Tn Ag) or(GalNAcα-Ser/Thr)); prostate-specific membrane antigen (PSMA); Receptortyrosine kinase-like orphan receptor 1 (ROR1); Fms-Like Tyrosine Kinase3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6;Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule(EPCAM); B7H3 (CD276); KIT (CD117); Interleukin-13 receptor subunitalpha-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin 11 receptor alpha(IL-11Ra); prostate stem cell antigen (PSCA); Protease Serine 21(Testisin or PRSS21); vascular endothelial growth factor receptor 2(VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growth factorreceptor beta (PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4);CD20; Folate receptor alpha; Receptor tyrosine-protein kinase ERBB2(Her2/neu); Mucin 1, cell surface associated (MUC1); epidermal growthfactor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostase;prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M);Ephrin B2; fibroblast activation protein alpha (FAP); insulin-likegrowth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX);Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2);glycoprotein 100 (gp100); oncogene fusion protein consisting ofbreakpoint cluster region (BCR) and Abelson murine leukemia viraloncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type-A receptor 2(EphA2); Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); gangliosideGM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5);high molecular weight-melanoma-associated antigen (HMWMAA); o-acetyl-GD2ganglioside (OAcGD2); Folate receptor beta; tumor endothelial marker 1(TEM1/CD248); tumor endothelial marker 7-related (TEM7R); claudin 6(CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupledreceptor class C group 5, member D (GPRC5D); chromosome X open readingframe 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK);Polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion ofgloboH glycoceramide (GloboH); mammary gland differentiation antigen(NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1(HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); Gprotein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locusK 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR Gamma AlternateReading Frame Protein (TARP); Wilms tumor protein (WT1); Cancer/testisantigen 1 (NY-ESO-1); Cancer/testis antigen 2 (LAGE-1a);Melanoma-associated antigen 1 (MAGE-A1); ETS translocation-variant gene6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); XAntigen Family, Member 1A (XAGE1); angiopoietin-binding cell surfacereceptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1);melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1;tumor protein p53 (p53); p53 mutant; prostein; surviving; telomerase;prostate carcinoma tumor antigen-1 (PCTA-1 or Galectin 8), melanomaantigen recognized by T cells 1 (MelanA or MART1); Rat sarcoma (Ras)mutant; human Telomerase reverse transcriptase (hTERT); sarcomatranslocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG(transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetylglucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3);Androgen receptor; Cyclin B1; v-myc avian myelocytomatosis viraloncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family MemberC (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 1B1(CYP1B1); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS orBrother of the Regulator of Imprinted Sites), Squamous Cell CarcinomaAntigen Recognized By T Cells 3 (SART3); Paired box protein Pax-5(PAX5); proacrosin binding protein sp32 (OY-TES1); lymphocyte-specificprotein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4);synovial sarcoma, X breakpoint 2 (SSX2); Receptor for Advanced GlycationEndproducts (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2(RU2); legumain; human papilloma virus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxyl esterase; heat shock protein 70-2mutated (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associatedimmunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor(FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily Amember 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-typelectin domain family 12 member A (CLEC12A); bone marrow stromal cellantigen 2 (BST2); EGF-like module-containing mucin-like hormonereceptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3);Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1(IGLL1).

CD19 CAR Constructs

Murine CD19 CAR constructs are described in PCT publication WO2012/079000, incorporated herein by reference, and the amino acidsequence of the murine CD19 CAR and scFv constructs are shown in Table 2below.

TABLE 2 Murine CD19 CAR Constructs CTL019 Full- SEQ IDMALPVTALLLPLALLLHAARPdiqmtqttsslsaslgdrvtiscrasqdiskylnw aa NO: 81yqqkpdgtvklliyhtsrlhsgvpsrfsgsgsgtdysltisnleqediatyfcqqgntlpytfgggtkleitggggsggggsggggsevklqesgpglvapsqslsvtctvsgvslpdygvswirqpprkglewlgviwgsettyynsalksrltiikdnsksqvflkmnslqtddtaiyycakhyyyggsyamdywgqgtsvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr CTL019 scFv SEQ IDDiqmtqttsslsaslgdrvtiscrasqdiskylnwyqqkpdgtvklliyhtsrlhs domain NO: 52gvpsrfsgsgsgtdysltisnleqediatyfcqqgntlpytfgggtkleitggggsggggsggggsevklqesgpglvapsgslsvtctvsgvslpdygvswirqpprkglewlgviwgsettyynsalksrltiikdnsksqvflkmnslqtddtaiyycakhyyyggsyamdywgqgtsvtvss mCAR1 scFv SEQ IDQVQLLESGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDG NO: 84DTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYSCARKTISSVVDFYFDYWGQGTTVTGGGSGGGSGGGSGGGSELVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQYNRYPYTSFFFTKLEIKRRS mCAR1 Full- SEQ IDQVQLLESGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDG aa NO: 85DTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYSCARKTISSVVDFYFDYWGQGTTVTGGGSGGGSGGGSGGGSELVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQYNRYPYTSFFFTKLEIKRRSKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFTIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR mCAR2 scFv SEQ IDDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHS NO: 86GVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSE mCAR2 CAR- SEQ IDDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHS aa NO: 87GVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSESKYGPPCPPCPMFWVLVVVGGVLACYSLLVTVAFTIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRL mCAR2 Full- SEQ IDDIQMTQTT SSLSASLGDR VTISCRASQD ISKYLNWYQQ KPDGTVKLLI aa NO: 88YHTSRLHSGV PSRFSGSGSG TDYSLTISNL EQEDIATYFC QQGNTLPYTFGGGTKLEITG STSGSGKPGS GEGSTKGEVK LQESGPGLVA PSQSLSVTCTVSGVSLPDYG VSWIRQPPRK GLEWLGVIWG SETTYYNSAL KSRLTIIKDNSKSQVFLKMN SLQTDDTAIY YCAKHYYYGG SYAMDYWGQG TSVTVSSESKYGPPCPPCPM FWVLVVVGGV LACYSLLVTVAFIIFWVKRG RKKLLYIFKQ PFMRPVQTTQ EEDGCSCRFE EEEGGCELRVKFSRSADAPA YQQGQNQLYN ELNLGRREEY DVLDKRRGRD PEMGGKPRRKNPQEGLYNEL QKDKMAEAYS EIGMKGERRR GKGHDGLYQG LSTATKDTYDALHMQALPPR LEGGGEGRGS LLTCGDVEEN PGPRMLLLVT SLLLCELPHPAFLLIPRKVC NGIGIGEFKD SLSINATNIK HFKNCTSISG DLHILPVAFRGDSFTHTPPL DPQELDILKT VKEITGFLLI QAWPENRTDL HAFENLEIIRGRTKQHGQFS LAVVSLNITS LGLRSLKEIS DGDVIISGNK NLCYANTINWKKLFGTSGQK TKIISNRGEN SCKATGQVCH ALCSPEGCWG PEPRDCVSCRNVSRGRECVD KCNLLEGEPR EFVENSECIQ CHPECLPQAM NITCTGRGPDNCIQCAHYID GPHCVKTCPA GVMGENNTLV WKYADAGHVC HLCHPNCTYGCTGPGLEGCP TNGPKIPSIA TGMVGALLLL LVVALGIGLF M mCAR3 scFv SEQ IDDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHS NO: 89GVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS mCAR3 Full- SEQ IDDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHS aa NO: 90GVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFTIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

CD19 CAR constructs containing humanized anti-CD19 scFv domains aredescribed in PCT publication WO 2014/153270, incorporated herein byreference.

In an embodiment, the antigen binding domain comprises an anti-CD19antibody, or fragment thereof, e.g., an scFv. For example, the antigenbinding domain comprises a variable heavy chain and a variable lightchain listed in Table 12. The linker sequence joining the variable heavyand variable light chains can be, e.g., any of the linker sequencesdescribed herein, or alternatively, can be GSTSGSGKPGSGEGSTKG (SEQ IDNO:45).

TABLE 12 Anti-CD 19 antibody binding domains CD19 huscFv1EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLTYHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLICTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYSSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS (SEQ ID NO: 24) CD19 huscFv2Eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyygsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss (SEQ ID NO: 25) CD19 huscFv3Qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyssslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik (SEQ ID NO: 26) CD19 huscFv4Qvqlqesqpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik (SEQ ID NO: 27) CD19 huscFv5Eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsggggsggggsqvqlgesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyssslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss (SEQ ID NO: 39) CD19 huscFv6Eivmtgspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsggggsggggsqvglgesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyygsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtivtvss (SEQ ID NO: 43) CD19 huscFv7Qvglgesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyssslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtivtvssggggsggggsggggsggggseivmtgspat1s1spgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik (SEQ ID NO: 46) CD19 huscFv8Qvglgesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyygsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtivtvssggggsggggsggggsggggseivmtgspat1s1spgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik (SEQ ID NO: 47) CD19 huscFv9Eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsggggsggggsqvqlgesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyynsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtivtvss (SEQ ID NO: 48) CD19 HuQvglqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyy scFv10nsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik (SEQ ID NO: 49) CD19 HuEivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgip scFv11arfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsggggsqvqlgesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyynsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss (SEQ ID NO: 50) CD19 HuQvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyy scFv12nsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggseivmtgspat1s1spgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik (SEQ ID NO: 51) CD19 muCTL019Diqmtqttsslsaslgdrvtiscrasqdiskylnwyqqkpdgtvklliyhtsrlhsgvpsrfsgsgsgtdysltisnleqediatyfcqqgntlpytfgggtkleitggggsggggsggggsevklqesgpglvapsqslsvtctvsgvslpdygvswirqpprkglewlgviwgsettyynsalksrltiikdnsksqvflkmnslqtddtaiyycakhyyyggsyamdywgqgtsvtvss (SEQ ID NO: 52) CD19 SSJ25-QVQLLESGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTN Cl VHYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYSCARKTISSVVDFYFDYWGQGTTV sequenceT (SEQ ID NO: 53) CD19 SSJ25-ELVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVP Cl VLDRFTGSGSGTDFTLTITNVQSKDLADYFYFCQYNRYPYTSGGGTKLEIKRRS (SEQ sequenceID NO: 54)

Any known CD19 CAR, e.g., the CD19 antigen binding domain of any knownCD19 CAR, in the art can be used in accordance with the presentdisclosure. For example, LG-740; CD19 CAR described in the U.S. Pat.Nos. 8,399,645; 7,446,190; Xu et al., LEUK LYMPHOMA. 201354(2):255-260(2012); Cruz et al., BLOOD 122(17):2965-2973 (2013);Brentjens et al., BLOOD, 118(18):4817-4828 (2011); Kochenderfer et al.,BLOOD 116(20):4099-102 (2010); Kochenderfer et al., BLOOD 122(25):4129-39(2013); and 16th Annu Meet Am Soc Gen Cell Ther (ASGCT) (May15-18, Salt Lake City) 2013, Abst 10.

In one embodiment, the antigen binding domain comprises one, two three(e.g., all three) heavy chain CDRs, HC CDR1, HC CDR2 and HC CDR3, froman antibody listed above, and/or one, two, three (e.g., all three) lightchain CDRs, LC CDR1, LC CDR2 and LC CDR3, from an antibody listed above.In one embodiment, the antigen binding domain comprises a heavy chainvariable region and/or a variable light chain region of an antibodylisted or described above.

In an embodiment, an antigen binding domain against CD22 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., Haso etal., BLOOD, 121(7): 1165-1174 (2013); Wayne et al., CLIN CANCER RES16(6): 1894-1903 (2010); Kato et al., LEUK RES 37(1):83-88 (2013);Creative BioMart (creativebiomart.net): MOM-18047-S(P).

In an embodiment, an antigen binding domain against CD20 is an antigenbinding portion, e.g., CDRs, of the anitbody Rituximab, Ofatumumab,Ocrelizumab, Veltuzumab, or GA101.

In an embodiment, an antigen binding domain against ROR1 is an antigenbinding portion, e.g., CDRs, of an antibody described in, e.g., Hudeceket al., CLIN CANCER RES 19(12):3153-3164 (2013); WO 2011159847; andUS20130101607.

Bispecific CARS

In an embodiment a multispecific antibody molecule is a bispecificantibody molecule. A bispecific antibody has specificity for no morethan two antigens. A bispecific antibody molecule is characterized by afirst immunoglobulin variable domain sequence which has bindingspecificity for a first epitope and a second immunoglobulin variabledomain sequence that has binding specificity for a second epitope. In anembodiment the first and second epitopes are on the same antigen, e.g.,the same protein (or subunit of a multimeric protein). In an embodimentthe first and second epitopes overlap. In an embodiment the first andsecond epitopes do not overlap. In an embodiment the first and secondepitopes are on different antigens, e.g., different proteins (ordifferent subunits of a multimeric protein). In an embodiment abispecific antibody molecule comprises a heavy chain variable domainsequence and a light chain variable domain sequence which have bindingspecificity for a first epitope and a heavy chain variable domainsequence and a light chain variable domain sequence which have bindingspecificity for a second epitope. In an embodiment a bispecific antibodymolecule comprises a half antibody having binding specificity for afirst epitope and a half antibody having binding specificity for asecond epitope. In an embodiment a bispecific antibody moleculecomprises a half antibody, or fragment thereof, having bindingspecificity for a first epitope and a half antibody, or fragmentthereof, having binding specificity for a second epitope. In anembodiment a bispecific antibody molecule comprises a scFv, or fragmentthereof, have binding specificity for a first epitope and a scFv, orfragment thereof, have binding specificity for a second epitope.

In certain embodiments, the antibody molecule is a multi-specific (e.g.,a bispecific or a trispecific) antibody molecule. Protocols forgenerating bispecific or heterodimeric antibody molecules, and variousconfigurations for bispecific antibody molecules, are described in,e.g., paragraphs 455-458 of WO2015/142675, filed Mar. 13, 2015, which isincorporated by reference in its entirety.

In one aspect, the bispecific antibody molecule is characterized by afirst immunoglobulin variable domain sequence, e.g., a scFv, which hasbinding specificity for CD19, e.g., comprises a scFv as describedherein, or comprises the light chain CDRs and/or heavy chain CDRs from ascFv described herein, and a second immunoglobulin variable domainsequence that has binding specificity for a second epitope on adifferent antigen.

Chimeric TCR

In one aspect, the antibodies and antibody fragments of the presentinvention (e.g., CD19 antibodies and fragments) can be grafted to one ormore constant domain of a T cell receptor (“TCR”) chain, for example, aTCR alpha or TCR beta chain, to create a chimeric TCR. Without beingbound by theory, it is believed that chimeric TCRs will signal throughthe TCR complex upon antigen binding. For example, an scFv as disclosedherein, can be grafted to the constant domain, e.g., at least a portionof the extracellular constant domain, the transmembrane domain and thecytoplasmic domain, of a TCR chain, for example, the TCR alpha chainand/or the TCR beta chain. As another example, an antibody fragment, forexample a VL domain as described herein, can be grafted to the constantdomain of a TCR alpha chain, and an antibody fragment, for example a VHdomain as described herein, can be grafted to the constant domain of aTCR beta chain (or alternatively, a VL domain may be grafted to theconstant domain of the TCR beta chain and a VH domain may be grafted toa TCR alpha chain). As another example, the CDRs of an antibody orantibody fragment may be grafted into a TCR alpha and/or beta chain tocreate a chimeric TCR. For example, the LCDRs disclosed herein may begrafted into the variable domain of a TCR alpha chain and the HCDRsdisclosed herein may be grafted to the variable domain of a TCR betachain, or vice versa. Such chimeric TCRs may be produced, e.g., bymethods known in the art (For example, Willemsen R A et al, Gene Therapy2000; 7: 1369-1377; Zhang T et al, Cancer Gene Ther 2004; 11: 487-496;Aggen et al, Gene Ther. 2012 April; 19(4):365-74).

Non Antibody Scaffolds

In embodiments, the antigen binding domain comprises a non-antibodyscaffold, e.g., a fibronectin, ankyrin, domain antibody, lipocalin,small modular immuno-pharmaceutical, maxybody, Protein A, or affilin.The non-antibody scaffold has the ability to bind to target antigen on acell. In embodiments, the antigen binding domain is a polypeptide orfragment thereof of a naturally occurring protein expressed on a cell.In some embodiments, the antigen binding domain comprises a non-antibodyscaffold. A wide variety of non-antibody scaffolds can be employed solong as the resulting polypeptide includes at least one binding regionwhich specifically binds to the target antigen on a target cell.

Non-antibody scaffolds include: fibronectin (Novartis, MA), ankyrin(Molecular Partners AG, Zurich, Switzerland), domain antibodies(Domantis, Ltd., Cambridge, Mass., and Ablynx nv, Zwijnaarde, Belgium),lipocalin (Pieris Proteolab AG, Freising, Germany), small modularimmuno-pharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, Wash.),maxybodies (Avidia, Inc., Mountain View, Calif.), Protein A (AffibodyAG, Sweden), and affilin (gamma-crystallin or ubiquitin) (Scil ProteinsGmbH, Halle, Germany).

In an embodiment the antigen binding domain comprises the extracellulardomain, or a counter-ligand binding fragment thereof, of molecule thatbinds a counterligand on the surface of a target cell.

Transmembrane Domain

In embodiments, a CAR described herein comprises a transmembrane domainthat is fused to an extracellular sequence, e.g., an extracellularrecognition element, which can comprise an antigen binding domain. In anembodiment, the transmembrane domain is one that naturally is associatedwith one of the domains in the CAR. In an embodiment, the transmembranedomain is one that is not naturally associated with one of the domainsin the CAR.

A transmembrane domain can include one or more additional amino acidsadjacent to the transmembrane region, e.g., one or more amino acidassociated with the extracellular region of the protein from which thetransmembrane was derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15amino acids of the extracellular region) and/or one or more additionalamino acids associated with the intracellular region of the protein fromwhich the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, 10 up to 15 amino acids of the intracellular region).

In embodiments, the transmembrane domain is one which minimizesinteractions with other elements, e.g., other transmembrane domains. Insome instances, the transmembrane domain minimizes binding of suchdomains to the transmembrane domains of the same or different surfacemembrane proteins, e.g., to minimize interactions with other members ofthe receptor complex. Suitable examples can be derived by selection ormodification of amino acid substitution of a known transmembrane domain.In an embodiment, the transmembrane domain is capable of promotinghomodimerization with another CAR on the cell surface.

The transmembrane domain may comprise a naturally occurring, or anon-naturally occurring synthetic sequence. Where naturally occurring,the transmembrane domain may be derived from any membrane-bound ortransmembrane protein.

Transmembrane regions suitable for use in molecules described herein maybe derived from any one or more of e.g., the alpha, beta or zeta chainof the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9,CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In someembodiments, a transmembrane domain may include at least thetransmembrane region(s) of, e.g., KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a,CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR),SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2Rgamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6,CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b,ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM (e.g.,CEACAM-1, CEACAM-3 and/or CEACAM-5), CRTAM, Ly9 (CD229), CD160 (BY55),PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150,IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKG2D, NKG2C, orCD19. In an embodiment the transmembrane domain is derived from CD8. Inan embodiment the transmembrane domain is derived from CD28. In oneaspect, the transmembrane domain is a transmembrane domain from thesequence provided as SEQ ID NO: 12 or SEQ ID NO: 42.

In an embodiment, a sequence, e.g., a hinge or spacer sequence, can bedisposed between a transmembrane domain and another sequence or domainto which it is fused. In embodiments, a variety of human hinges (aka“spacers”) can be employed as well, e.g., including but not limited tothe human Ig (immunoglobulin) hinge. Optionally, a short oligo- orpolypeptide linker, between 2 and 10 amino acids in length may form thelinkage between the transmembrane domain and another domain, e.g., anintracellular signaling domain or costimulatory domain, of a CAR. Aglycine-serine doublet provides a particularly suitable linker. In oneaspect, the hinge or spacer is the amino acid sequence provided as SEQID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. In one aspect, the hinge orspacer comprises a KIR2DS2 hinge.

In an embodiment, the transmembrane domain may be a non-naturallyoccurring sequence, in which case can comprise predominantly hydrophobicresidues such as leucine and valine. In an embodiment, a triplet ofphenylalanine, tryptophan and valine will be found at each end of atransmembrane domain.

Optionally, a short oligo- or polypeptide linker, between 2 and 10 aminoacids in length may form the linkage between the transmembrane domainand the cytoplasmic region of the CAR. A glycine-serine doublet providesa particularly suitable linker. For example, in one aspect, the linkercomprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO:10). In someembodiments, the linker is encoded by a nucleotide sequence ofGGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO:11).

Cytoplasmic Domain

The cytoplasmic domain or region of the CAR includes an intracellularsignaling domain. An intracellular signaling domain is generallyresponsible for activation of at least one of the normal effectorfunctions of the immune cell in which the CAR has been introduced.

Examples of intracellular signaling domains for use in the CAR of theinvention include the cytoplasmic sequences of the T cell receptor (TCR)and co-receptors that act in concert to initiate signal transductionfollowing antigen receptor engagement, as well as any derivative orvariant of these sequences and any recombinant sequence that has thesame functional capability.

It is known that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondaryand/or costimulatory signal is also required. Thus, T cell activationcan be said to be mediated by two distinct classes of cytoplasmicsignaling sequences: those that initiate antigen-dependent primaryactivation through the TCR (primary intracellular signaling domains) andthose that act in an antigen-independent manner to provide a secondaryor costimulatory signal (secondary cytoplasmic domain, e.g., acostimulatory domain).

Primary Signaling Domain

A primary signaling domain regulates primary activation of the TCRcomplex either in a stimulatory way, or in an inhibitory way. Primaryintracellular signaling domains that act in a stimulatory manner maycontain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs.

Examples of ITAM containing primary intracellular signaling domains thatare of particular use in the invention include those of TCR zeta, FcRgamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a,CD79b, CD278 (also known as “ICOS”), FccRI, DAP10, DAP12, and CD66d. Inone embodiment, a CAR of the invention comprises an intracellularsignaling domain, e.g., a primary signaling domain of CD3-zeta, e.g., aCD3-zeta sequence described herein.

In one embodiment, a primary signaling domain comprises a modified ITAMdomain, e.g., a mutated ITAM domain which has altered (e.g., increasedor decreased) activity as compared to the native ITAM domain. In oneembodiment, a primary signaling domain comprises a modifiedITAM-containing primary intracellular signaling domain, e.g., anoptimized and/or truncated ITAM-containing primary intracellularsignaling domain. In an embodiment, a primary signaling domain comprisesone, two, three, four or more ITAM motifs. Further examples of moleculescontaining a primary intracellular signaling domain that are ofparticular use in the invention include those of DAP10, DAP12, and CD32.

A primary intracellular signaling domain comprises a functionalfragment, or analog, of a primary stimulatory molecule (e.g., CD3zeta-GenBank Acc. No. BAG36664.1). The primary intracellular signalingdomain can comprise the entire intracellular region or a fragment of theintracellular region which is sufficient for generation of anintracellular signal when an antigen binding domain to which it is fusedbinds cognate antigen. In embodiments the primary intracellularsignaling domain has at least 70, 75, 80, 85, 90, 95, 98, or 99%sequence identity with the entire intracellular region, or a fragment ofthe intracellular region which is sufficient for generation of anintracellular signal, of a naturally occurring primary stimulatorymolecule, e.g., a human (GenBank Acc No. BAG36664.1), or othermammalian, e.g., a nonhuman species, e.g., rodent, monkey, ape or murineintracellular primary stimulatory molecule. In embodiments the primaryintracellular signaling domain has at least 70, 75, 80, 85, 90, 95, 98,or 99% sequence identity with SEQ ID NO: 18 or SEQ ID NO: 20.

In embodiments, the primary intracellular signaling domain, has at least70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity with, or differs byno more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residuesfrom the corresponding residues of the entire intracellular region, or afragment of the intracellular region which is sufficient for generationof an intracellular signal, of a naturally occurring human primarystimulatory molecule, e.g., a naturally occurring human primarystimulatory molecule disclosed herein.

Costimulatory Signaling Domain

The intracellular signalling domain of the CAR can comprise the CD3-zetasignalling domain by itself or it can be combined with any other desiredintracellular signalling domain(s) useful in the context of a CAR of theinvention. For example, the intracellular signalling domain of the CARcan comprise a CD3 zeta chain portion and a costimulatory signalingdomain. The costimulatory signaling domain refers to a portion of theCAR comprising the intracellular domain of a costimulatory molecule. Inone embodiment, the intracellular domain is designed to comprise thesignaling domain of CD3-zeta and the signaling domain of CD28. In oneaspect, the intracellular domain is designed to comprise the signalingdomain of CD3-zeta and the signaling domain of ICOS.

A costimulatory molecule is a cell surface molecule other than anantigen receptor or its ligands that is required for an efficientresponse of lymphocytes to an antigen. Examples of such moleculesinclude MHC class I molecule, TNF receptor proteins, Immunoglobulin-likeproteins, cytokine receptors, integrins, signaling lymphocyticactivation molecules (SLAM proteins), activating NK cell receptors,BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40,CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS(CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta,IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6,VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM,CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D,NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84,CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3),BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a,and a ligand that specifically binds with CD83, and the like. Forexample, CD27 costimulation has been demonstrated to enhance expansion,effector function, and survival of human CAR-expressing cell (e.g., Tcell, NK cell) cells in vitro and augments human T cell persistence andantitumor activity in vivo (Song et al. BLOOD. 2012; 119(3):696-706).Further examples of such costimulatory molecules include CDS, ICAM-1,GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46,CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha,ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD,CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c,ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1(CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A,Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162),LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKG2D and NKG2C.

The intracellular signaling sequences within the cytoplasmic portion ofthe CAR of the invention may be linked to each other in a random orspecified order. Optionally, a short oligo- or polypeptide linker, forexample, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or10 amino acids) in length may form the linkage between intracellularsignaling sequence. In one embodiment, a glycine-serine doublet can beused as a suitable linker. In one embodiment, a single amino acid, e.g.,an alanine, a glycine, can be used as a suitable linker.

In one aspect, the intracellular signaling domain is designed tocomprise two or more, e.g., 2, 3, 4, 5, or more, costimulatory signalingdomains. In an embodiment, the two or more, e.g., 2, 3, 4, 5, or more,costimulatory signaling domains, are separated by a linker molecule,e.g., a linker molecule described herein. In one embodiment, theintracellular signaling domain comprises two costimulatory signalingdomains. In some embodiments, the linker molecule is a glycine residue.In some embodiments, the linker is an alanine residue.

A costimulatory domain comprises a functional fragment, or analog, of acostimulatory molecule (e.g., ICOS, CD28, or 4-1BB). It can comprise theentire intracellular region or a fragment of the intracellular regionwhich is sufficient for generation of an intracellular signal, e.g.,when an antigen binding domain to which it is fused binds cognateantigen. In embodiments the costimulatory domain has at least 70%, 75%,80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the entireintracellular region, or a fragment of the intracellular region which issufficient for generation of an intracellular signal, of a naturallyoccurring costimulatory molecule as described herein, e.g., a human, orother mammalian, e.g., a nonhuman species, e.g., rodent, monkey, ape ormurine intracellular costimulatory molecule. In embodiments thecostimulatory domain has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or99% sequence identity with SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 40,or SEQ ID NO: 44.

In embodiments the costimulatory signaling domain, has at least 70, 75,80, 85, 90, 95, 96, 97, 98, or 99% identity with, or differs by no morethan 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residues from thecorresponding residues of the entire intracellular region, or a fragmentof the intracellular region which is sufficient for generation of anintracellular signal, of, a naturally occurring human costimulatorymolecule, e.g., a naturally occurring human costimulatory moleculedisclosed herein.

Any of the CARs described herein can include one or more of thecomponents listed in Table 11.

TABLE 11 Sequences of various components of CAR(aa—amino acids, na—nucleic acidsthat encodes the corresponding protein) SEQ ID NO description Sequence 1EF-1 CGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCC promoterCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA 2 Leader (aa)MALPVTALLLPLALLLHAARP 3 Leader (na)ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCATGC CGCTAGACCC 4CD 8 hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (aa) 5CD8 hinge ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCG (na)CAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT 6 Ig4 hingeESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV (aa)QFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGKM 7Ig4 hinge GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGG (na)GCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATG 8 IgD hingeRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQ (aa)EERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLL NASRSLEVSYVTDH 9IgD hinge AGGTGGCCCGAAAGTCCCAAGGCCCAGGCATCTAGTGTTCCTACTGCACAGC (na)CCCAGGCAGAAGGCAGCCTAGCCAAAGCTACTACTGCACCTGCCACTACGCGCAATACTGGCCGTGGCGGGGAGGAGAAGAAAAAGGAGAAAGAGAAAGAAGAACAGGAAGAGAGGGAGACCAAGACCCCTGAATGTCCATCCCATACCCAGCCGCTGGGCGTCTATCTCTTGACTCCCGCAGTACAGGACTTGTGGCTTAGAGATAAGGCCACCTTTACATGTTTCGTCGTGGGCTCTGACCTGAAGGATGCCCATTTGACTTGGGAGGTTGCCGGAAAGGTACCCACAGGGGGGGTTGAGGAAGGGTTGCTGGAGCGCCATTCCAATGGCTCTCAGAGCCAGCACTCAAGACTCACCCTTCCGAGATCCCTGTGGAACGCCGGGACCTCTGTCACATGTACTCTAAATCATCCTAGCCTGCCCCCACAGCGTCTGATGGCCCTTAGAGAGCCAGCCGCCCAGGCACCAGTTAAGCTTAGCCTGAATCTGCTCGCCAGTAGTGATCCCCCAGAGGCCGCCAGCTGGCTCTTATGCGAAGTGTCCGGCTTTAGCCCGCCCAACATCTTGCTCATGTGGCTGGAGGACCAGCGAGAAGTGAACACCAGCGGCTTCGCTCCAGCCCGGCCCCCACCCCAGCCGGGTTCTACCACATTCTGGGCCTGGAGTGTCTTAAGGGTCCCAGCACCACCTAGCCCCCAGCCAGCCACATACACCTGTGTTGTGTCCCATGAAGATAGCAGGACCCTGCTAAATGCTTCTAGGAGTCTGGAGGTTTCCTACGTGACTGACCATT 10 GS GGGGSGGGGS hinge/linker (aa) 11 GSGGTGGCGGAGGTTCTGGAGGTGGAGGTTCC hinge/linker (na) 12 CD8TM (aa)IYIWAPLAGTCGVLLLSLVITLYC 13 CD8 TM (na)ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC 14 4-1BB KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELintracellular domain (aa) 15 4-1BBAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGAC intracellularCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAG domain (na)AAGAAGAAGGAGGATGTGAACTG 16 CD27 (aa)QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP 17 CD 27 (na)AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC 18 CD3-zetaRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK (aa)NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR 19CD3-zeta AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCA (na)GAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC 20 CD3-zetaRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK (aa)NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR 21CD3-zeta AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCA (na)GAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC 22 linker GGGGS 23 linkerGGTGGCGGAGGTTCTGGAGGTGGAGGTTCC 28 linker(Gly-Gly-Gly-Ser)_(n), where n = 1-10 29 linker (Gly4 Ser)4 30 linker(Gly4 Ser)3 31 linker (Gly3Ser) 32 polyA A₂₀₀₀ 33 polyA A₁₅₀ 34 polyAA₅₀₀₀ 35 polyT T₁₀₀ 36 polyT T₅₀₀₀ 37 polyA A₆₄ 38 polyA A₄₀₀

Combination of CARs

In one aspect, the CAR-expressing cell described herein can furthercomprise a second CAR, e.g., a second CAR that includes a differentantigen binding domain, e.g., to the same target or a different target(e.g., a target other than a cancer associated antigen described hereinor a different cancer associated antigen described herein, e.g., CD19,CD33, CLL-1, CD34, FLT3, or folate receptor beta). In one embodiment,the second CAR includes an antigen binding domain to a target expressedthe same cancer cell type as the cancer associated antigen. In oneembodiment, the CAR-expressing cell comprises a first CAR that targets afirst antigen and includes an intracellular signaling domain having acostimulatory signaling domain but not a primary signaling domain, and asecond CAR that targets a second, different, antigen and includes anintracellular signaling domain having a primary signaling domain but nota costimulatory signaling domain. While not wishing to be bound bytheory, placement of a costimulatory signaling domain, e.g., 4-1BB,CD28, ICOS, CD27 or OX-40, onto the first CAR, and the primary signalingdomain, e.g., CD3 zeta, on the second CAR can limit the CAR activity tocells where both targets are expressed. In one embodiment, the CARexpressing cell comprises a first cancer associated antigen CAR thatincludes an antigen binding domain that binds a target antigen describedherein, a transmembrane domain and a costimulatory domain and a secondCAR that targets a different target antigen (e.g., an antigen expressedon that same cancer cell type as the first target antigen) and includesan antigen binding domain, a transmembrane domain and a primarysignaling domain. In another embodiment, the CAR expressing cellcomprises a first CAR that includes an antigen binding domain that bindsa target antigen described herein, a transmembrane domain and a primarysignaling domain and a second CAR that targets an antigen other than thefirst target antigen (e.g., an antigen expressed on the same cancer celltype as the first target antigen) and includes an antigen binding domainto the antigen, a transmembrane domain and a costimulatory signalingdomain.

In one embodiment, the CAR-expressing cell comprises a CAR describedherein (e.g., a CD19 CAR) and an inhibitory CAR. In one embodiment, theinhibitory CAR comprises an antigen binding domain that binds an antigenfound on normal cells but not cancer cells, e.g., normal cells that alsoexpress CLL. In one embodiment, the inhibitory CAR comprises the antigenbinding domain, a transmembrane domain and an intracellular domain of aninhibitory molecule. For example, the intracellular domain of theinhibitory CAR can be an intracellular domain of PD1, PD-L1, PD-L2,CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3,VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II,GAL9, adenosine, and TGFR (e.g., TGFRbeta).

In one embodiment, when the CAR-expressing cell comprises two or moredifferent CARs, the antigen binding domains of the different CARs can besuch that the antigen binding domains do not interact with one another.For example, a cell expressing a first and second CAR can have anantigen binding domain of the first CAR, e.g., as a fragment, e.g., anscFv, that does not form an association with the antigen binding domainof the second CAR, e.g., the antigen binding domain of the second CAR isa VHH.

In some embodiments, when present on the surface of a cell, binding ofthe antigen binding domain of the first CAR to its cognate antigen isnot substantially reduced by the presence of the second CAR. In someembodiments, binding of the antigen binding domain of the first CAR toits cognate antigen in the presence of the second CAR is 85%, 90%, 95%,96%, 97%, 98% or 99% of binding of the antigen binding domain of thefirst CAR to its cognate antigen in the absence of the second CAR.

In some embodiments, when present on the surface of a cell, the antigenbinding domains of the first CAR said second CAR, associate with oneanother less than if both were scFv antigen binding domains. In someembodiments, the antigen binding domains of the first CAR and the secondCAR, associate with one another 85%, 90%, 95%, 96%, 97%, 98% or 99% lessthan if both were scFv antigen binding domains.

CAR-Expressing Cells

The CARs described herein are expressed on cells, e.g., immune effectorcells, e.g., T cells. For example, a nucleic acid construct of a CARdescribed herein is transduced to a T cell. In embodiments, the cellsexpressing the CARs described herein are an in vitro transcribed RNA CART cell.

Sources of Cells, e.g., T Cells

Prior to expansion and genetic modification or other modification, asource of cells, e.g., immune effector cells, e.g., T cells or NK cells,can be obtained from a subject. Examples of subjects include humans,monkeys, chimpanzees, dogs, cats, mice, rats, and transgenic speciesthereof. T cells can be obtained from a number of sources, includingperipheral blood mononuclear cells, bone marrow, lymph node tissue, cordblood, thymus tissue, tissue from a site of infection, ascites, pleuraleffusion, spleen tissue, and tumors. In some embodiments, the cellsobtained as described in this section are subjected to an assaydescribed herein, e.g., one or more biomarkers are assayed.

In certain aspects of the present disclosure, immune effector cells,e.g., T cells or NK cells, can be obtained from a unit of bloodcollected from a subject using any number of techniques known to theskilled artisan, such as Ficoll™ separation. In one aspect, cells fromthe circulating blood of an individual are obtained by apheresis. Theapheresis product typically contains lymphocytes, including T cells,monocytes, granulocytes, B cells, other nucleated white blood cells, redblood cells, and platelets. In one aspect, the cells collected byapheresis may be washed to remove the plasma fraction and, optionally,to place the cells in an appropriate buffer or media for subsequentprocessing steps. In one embodiment, the cells are washed with phosphatebuffered saline (PBS). In an alternative embodiment, the wash solutionlacks calcium and may lack magnesium or may lack many if not alldivalent cations.

Initial activation steps in the absence of calcium can lead to magnifiedactivation. As those of ordinary skill in the art would readilyappreciate a washing step may be accomplished by methods known to thosein the art, such as by using a semi-automated “flow-through” centrifuge(for example, the Cobe 2991 cell processor, the Baxter CytoMate, or theHaemonetics Cell Saver 5) according to the manufacturer's instructions.After washing, the cells may be resuspended in a variety ofbiocompatible buffers, such as, for example, Ca-free, Mg-free PBS,PlasmaLyte A, or other saline solution with or without buffer.Alternatively, the undesirable components of the apheresis sample may beremoved and the cells directly resuspended in culture media.

It is recognized that the methods of the application can utilize culturemedia conditions comprising 5% or less, for example 2%, human AB serum,and employ known culture media conditions and compositions, for examplethose described in Smith et al., “Ex vivo expansion of human T cells foradoptive immunotherapy using the novel Xeno-free CTS Immune Cell SerumReplacement” Clinical & Translational Immunology (2015) 4, e31;doi:10.1038/cti.2014.31.

In one aspect, T cells are isolated from peripheral blood lymphocytes bylysing the red blood cells and depleting the monocytes, for example, bycentrifugation through a PERCOLL™ gradient or by counterflow centrifugalelutriation.

The methods described herein can include, e.g., selection of a specificsubpopulation of immune effector cells, e.g., T cells, that are a Tregulatory cell-depleted population, CD25+ depleted cells, using, e.g.,a negative selection technique, e.g., described herein. In embodiments,the population of T regulatory depleted cells contains less than 30%,25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells.

In one embodiment, T regulatory cells, e.g., CD25+ T cells, are removedfrom the population using an anti-CD25 antibody, or fragment thereof, ora CD25-binding ligand, e.g., IL-2. In one embodiment, the anti-CD25antibody, or fragment thereof, or CD25-binding ligand is conjugated to asubstrate, e.g., a bead, or is otherwise coated on a substrate, e.g., abead. In one embodiment, the anti-CD25 antibody, or fragment thereof, isconjugated to a substrate as described herein.

In one embodiment, the T regulatory cells, e.g., CD25+ T cells, areremoved from the population using CD25 depletion reagent from Miltenyi™.In one embodiment, the ratio of cells to CD25 depletion reagent is 1e7cells to 20 uL, or 1e7 cells to15 uL, or 1e7 cells to 10 uL, or 1e7cells to 5 uL, or 1e7 cells to 2.5 uL, or 1e7 cells to 1.25 uL. In oneembodiment, e.g., for T regulatory cells, e.g., CD25+ depletion, greaterthan 500 million cells/ml is used. In a further aspect, a concentrationof cells of 600, 700, 800, or 900 million cells/ml is used.

In one embodiment, the population of immune effector cells to bedepleted includes about 6×10⁹ CD25+ T cells. In other aspects, thepopulation of immune effector cells to be depleted include about 1×10⁹to 1×10¹⁰ CD25+ T cell, and any integer value in between. In oneembodiment, the resulting population T regulatory depleted cells has2×10⁹ T regulatory cells, e.g., CD25+ cells, or less (e.g., 1×10⁹,5×10⁸, 1×10⁸, 5×10⁷, 1×10⁷, or less CD25+ cells).

In one embodiment, the T regulatory cells, e.g., CD25+ cells, areremoved from the population using the CliniMAC system with a depletiontubing set, such as, e.g., tubing 162-01. In one embodiment, theCliniMAC system is run on a depletion setting such as, e.g.,DEPLETION2.1.

Without wishing to be bound by a particular theory, decreasing the levelof negative regulators of immune cells (e.g., decreasing the number ofunwanted immune cells, e.g., T_(REG) cells), in a subject prior toapheresis or during manufacturing of a CAR-expressing cell product canreduce the risk of subject relapse. In an embodiment, a patient ispre-treated with one or more therapies that reduce T_(REG) cells priorto collection of cells for CAR-expressing cell (e.g., T cell, NK cell)product manufacturing, thereby reducing the risk of patient relapse toCAR-expressing cell (e.g., T cell, NK cell) treatment (e.g., CTL019treatment). Methods of depleting T_(REG) cells are known in the art.Methods of decreasing T_(REG) cells include, but are not limited to,cyclophosphamide, anti-GITR antibody, CD25-depletion, and combinationsthereof.

In some embodiments, the manufacturing methods comprise reducing thenumber of (e.g., depleting) T_(REG) cells prior to manufacturing of theCAR-expressing cell. For example, manufacturing methods comprisecontacting the sample, e.g., the apheresis sample, with an anti-GITRantibody and/or an anti-CD25 antibody (or fragment thereof, or aCD25-binding ligand), e.g., to deplete T_(REG) cells prior tomanufacturing of the CAR-expressing cell (e.g., T cell, NK cell)product.

In an embodiment, a patient is pre-treated with cyclophosphamide priorto collection of cells for CAR-expressing cell (e.g., T cell, NK cell)product manufacturing, thereby reducing the risk of patient relapse toCAR-expressing cell treatment (e.g., CTL019 treatment). In anembodiment, a patient is pre-treated with an anti-GITR antibody prior tocollection of cells for CAR-expressing cell (e.g., T cell, NK cell)product manufacturing, thereby reducing the risk of patient relapse toCAR-expressing cell treatment (e.g., CTL019 treatment).

In an embodiment, the CAR-expressing cell (e.g., T cell, NK cell)manufacturing process is modified to deplete T_(REG) cells prior tomanufacturing of the CAR-expressing cell (e.g., T cell, NK cell) product(e.g., a CTL019 product). In an embodiment, CD25-depletion is used todeplete T_(REG) cells prior to manufacturing of the CAR-expressing cell(e.g., T cell, NK cell) product (e.g., a CTL019 product).

The methods described herein can include more than one selection step,e.g., more than one depletion step. Enrichment of a T cell population bynegative selection can be accomplished, e.g., with a combination ofantibodies directed to surface markers unique to the negatively selectedcells. One method is cell sorting and/or selection via negative magneticimmunoadherence or flow cytometry that uses a cocktail of monoclonalantibodies directed to cell surface markers present on the cellsnegatively selected. For example, to enrich for CD4+ cells by negativeselection, a monoclonal antibody cocktail can include antibodies toCD14, CD20, CD11b, CD16, HLA-DR, and CD8.

The methods described herein can further include removing cells from thepopulation which express a tumor antigen, e.g., a tumor antigen thatdoes not comprise CD25, e.g., CD19, CD30, CD38, CD123, CD20, CD14 orCD11b, to thereby provide a population of T regulatory depleted, e.g.,CD25+ depleted, and tumor antigen depleted cells that are suitable forexpression of a CAR, e.g., a CAR described herein. In one embodiment,tumor antigen expressing cells are removed simultaneously with the Tregulatory, e.g., CD25+ cells. For example, an anti-CD25 antibody, orfragment thereof, and an anti-tumor antigen antibody, or fragmentthereof, can be attached to the same substrate, e.g., bead, which can beused to remove the cells or an anti-CD25 antibody, or fragment thereof,or the anti-tumor antigen antibody, or fragment thereof, can be attachedto separate beads, a mixture of which can be used to remove the cells.In other embodiments, the removal of T regulatory cells, e.g., CD25+cells, and the removal of the tumor antigen expressing cells issequential, and can occur, e.g., in either order.

Also provided are methods that include removing cells from thepopulation which express a check point inhibitor, e.g., a check pointinhibitor described herein, e.g., one or more of PD1+ cells, LAG3+cells, and TIM3+ cells, to thereby provide a population of T regulatorydepleted, e.g., CD25+ depleted cells, and check point inhibitor depletedcells, e.g., PD1+, LAG3+ and/or TIM3+ depleted cells. Exemplary checkpoint inhibitors include B7-H1, B&-1, CD160, P1H, 2B4, PD1, TIM3, CEACAM(e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, TIGIT, CTLA-4, BTLAand LAIR1. In one embodiment, check point inhibitor expressing cells areremoved simultaneously with the T regulatory, e.g., CD25+ cells. Forexample, an anti-CD25 antibody, or fragment thereof, and an anti-checkpoint inhibitor antibody, or fragment thereof, can be attached to thesame bead which can be used to remove the cells, or an anti-CD25antibody, or fragment thereof, and the anti-check point inhibitorantibody, or fragment there, can be attached to separate beads, amixture of which can be used to remove the cells. In other embodiments,the removal of T regulatory cells, e.g., CD25+ cells, and the removal ofthe check point inhibitor expressing cells is sequential, and can occur,e.g., in either order.

Methods described herein can include a positive selection step. Forexample, T cells can isolated by incubation with anti-CD3/anti-CD28(e.g., 3×28)-conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, fora time period sufficient for positive selection of the desired T cells.In one embodiment, the time period is about 30 minutes. In a furtherembodiment, the time period ranges from 30 minutes to 36 hours or longerand all integer values there between. In a further embodiment, the timeperiod is at least 1, 2, 3, 4, 5, or 6 hours. In yet another embodiment,the time period is 10 to 24 hours, e.g., 24 hours. Longer incubationtimes may be used to isolate T cells in any situation where there arefew T cells as compared to other cell types, such in isolating tumorinfiltrating lymphocytes (TIL) from tumor tissue or fromimmunocompromised individuals. Further, use of longer incubation timescan increase the efficiency of capture of CD8+ T cells. Thus, by simplyshortening or lengthening the time T cells are allowed to bind to theCD3/CD28 beads and/or by increasing or decreasing the ratio of beads toT cells (as described further herein), subpopulations of T cells can bepreferentially selected for or against at culture initiation or at othertime points during the process. Additionally, by increasing ordecreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on thebeads or other surface, subpopulations of T cells can be preferentiallyselected for or against at culture initiation or at other desired timepoints.

In one embodiment, a T cell population can be selected that expressesone or more of IFN-γ, TNFα, IL-17A, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9,IL-21, CCL20,GM-CSF, IL-10, IL-13, granzyme B, and perforin, or otherappropriate molecules, e.g., other cytokines. Methods for screening forcell expression can be determined, e.g., by the methods described in PCTPublication No.: WO 2013/126712. In an embodiment, the T cell populationexpresses cytokine CCL20, IL-17a, IL-6, and combinations thereof.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain aspects, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (e.g., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one aspect, a concentrationof 10 billion cells/ml, 9 billion/ml, 8 billion/ml, 7 billion/ml, 6billion/ml, or 5 billion/ml is used. In one aspect, a concentration of 1billion cells/ml is used. In yet one aspect, a concentration of cellsfrom 75, 80, 85, 90, 95, or 100 million cells/ml is used. In furtheraspects, concentrations of 125 or 150 million cells/ml can be used.

Using high concentrations can result in increased cell yield, cellactivation, and cell expansion. Further, use of high cell concentrationsallows more efficient capture of cells that may weakly express targetantigens of interest, such as CD28-negative T cells, or from sampleswhere there are many tumor cells present (e.g., leukemic blood, tumortissue, etc.). Such populations of cells may have therapeutic value andwould be desirable to obtain. For example, using high concentration ofcells allows more efficient selection of CD8+ T cells that normally haveweaker CD28 expression.

In a related aspect, it may be desirable to use lower concentrations ofcells. By significantly diluting the mixture of T cells and surface(e.g., particles such as beads), interactions between the particles andcells is minimized. This selects for cells that express high amounts ofdesired antigens to be bound to the particles. For example, CD4+ T cellsexpress higher levels of CD28 and are more efficiently captured thanCD8+ T cells in dilute concentrations. In one aspect, the concentrationof cells used is 5×10⁶/ml. In other aspects, the concentration used canbe from about 1×10⁵/ml to 1×10⁶/ml, and any integer value in between.

In other aspects, the cells may be incubated on a rotator for varyinglengths of time at varying speeds at either 2-10° C. or at roomtemperature.

T cells for stimulation can also be frozen after a washing step. Wishingnot to be bound by theory, the freeze and subsequent thaw step providesa more uniform product by removing granulocytes and to some extentmonocytes in the cell population. After the washing step that removesplasma and platelets, the cells may be suspended in a freezing solution.While many freezing solutions and parameters are known in the art andwill be useful in this context, one method involves using PBS containing20% DMSO and 8% human serum albumin, or culture media containing 10%Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitablecell freezing media containing for example, Hespan and PlasmaLyte A, thecells then are frozen to −80° C. at a rate of 1° per minute and storedin the vapor phase of a liquid nitrogen storage tank. Other methods ofcontrolled freezing may be used as well as uncontrolled freezingimmediately at −20° C. or in liquid nitrogen.

In certain aspects, cryopreserved cells are thawed and washed asdescribed herein and allowed to rest for one hour at room temperatureprior to activation using the methods of the present invention.

Also contemplated in the context of the invention is the collection ofblood samples or apheresis product from a subject at a time period priorto when the expanded cells as described herein might be needed. As such,the source of the cells to be expanded can be collected at any timepoint necessary, and desired cells, such as T cells, isolated and frozenfor later use in immune effector cell therapy for any number of diseasesor conditions that would benefit from immune effector cell therapy, suchas those described herein. In one aspect a blood sample or an apheresisis taken from a generally healthy subject. In certain aspects, a bloodsample or an apheresis is taken from a generally healthy subject who isat risk of developing a disease, but who has not yet developed adisease, and the cells of interest are isolated and frozen for lateruse. In certain aspects, the T cells may be expanded, frozen, and usedat a later time. In certain aspects, samples are collected from apatient shortly after diagnosis of a particular disease as describedherein but prior to any treatments. In a further aspect, the cells areisolated from a blood sample or an apheresis from a subject prior to anynumber of relevant treatment modalities, including but not limited totreatment with agents such as natalizumab, efalizumab, antiviral agents,chemotherapy, radiation, immunosuppressive agents, such as cyclosporin,azathioprine, methotrexate, mycophenolate, and FK506, antibodies, orother immunoablative agents such as CAMPATH, anti-CD3 antibodies,cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid,steroids, FR901228, and irradiation.

In a further aspect of the present invention, T cells are obtained froma patient directly following treatment that leaves the subject withfunctional T cells. In this regard, it has been observed that followingcertain cancer treatments, in particular treatments with drugs thatdamage the immune system, shortly after treatment during the period whenpatients would normally be recovering from the treatment, the quality ofT cells obtained may be optimal or improved for their ability to expandex vivo. Likewise, following ex vivo manipulation using the methodsdescribed herein, these cells may be in a preferred state for enhancedengraftment and in vivo expansion. Thus, it is contemplated within thecontext of the present invention to collect blood cells, including Tcells, dendritic cells, or other cells of the hematopoietic lineage,during this recovery phase. Further, in certain aspects, mobilization(for example, mobilization with GM-CSF) and conditioning regimens can beused to create a condition in a subject wherein repopulation,recirculation, regeneration, and/or expansion of particular cell typesis favored, especially during a defined window of time followingtherapy. Illustrative cell types include T cells, B cells, dendriticcells, and other cells of the immune system.

In one embodiment, the immune effector cells expressing a CAR molecule,e.g., a CAR molecule described herein, are obtained from a subject thathas received a low, immune enhancing dose of an mTOR inhibitor. In anembodiment, the population of immune effector cells, e.g., T cells, tobe engineered to express a CAR, are harvested after a sufficient time,or after sufficient dosing of the low, immune enhancing, dose of an mTORinhibitor, such that the level of PD1 negative immune effector cells,e.g., T cells, or the ratio of PD1 negative immune effector cells, e.g.,T cells/PD1 positive immune effector cells, e.g., T cells, in thesubject or harvested from the subject has been, at least transiently,increased.

In other embodiments, population of immune effector cells, e.g., Tcells, which have, or will be engineered to express a CAR, can betreated ex vivo by contact with an amount of an mTOR inhibitor thatincreases the number of PD1 negative immune effector cells, e.g., Tcells or increases the ratio of PD1 negative immune effector cells,e.g., T cells/PD1 positive immune effector cells, e.g., T cells.

In one embodiment, a T cell population is diacylglycerol kinase(DGK)-deficient. DGK-deficient cells include cells that do not expressDGK RNA or protein, or have reduced or inhibited DGK activity.DGK-deficient cells can be generated by genetic approaches, e.g.,administering RNA-interfering agents, e.g., siRNA, shRNA, miRNA, toreduce or prevent DGK expression. Alternatively, DGK-deficient cells canbe generated by treatment with DGK inhibitors described herein.

In one embodiment, a T cell population is Ikaros-deficient.Ikaros-deficient cells include cells that do not express Ikaros RNA orprotein, or have reduced or inhibited Ikaros activity, Ikaros-deficientcells can be generated by genetic approaches, e.g., administeringRNA-interfering agents, e.g., siRNA, shRNA, miRNA, to reduce or preventIkaros expression. Alternatively, Ikaros-deficient cells can begenerated by treatment with Ikaros inhibitors, e.g., lenalidomide.

In embodiments, a T cell population is DGK-deficient andIkaros-deficient, e.g., does not express DGK and Ikaros, or has reducedor inhibited DGK and Ikaros activity. Such DGK and Ikaros-deficientcells can be generated by any of the methods described herein.

Allogeneic CAR

In embodiments described herein, the immune effector cell can be anallogeneic immune effector cell, e.g., T cell. For example, the cell canbe an allogeneic T cell, e.g., an allogeneic T cell lacking expressionof a functional T cell receptor (TCR) and/or human leukocyte antigen(HLA), e.g., HLA class I and/or HLA class II.

A T cell lacking a functional TCR can be, e.g., engineered such that itdoes not express any functional TCR on its surface, engineered such thatit does not express one or more subunits that comprise a functional TCR(e.g., engineered such that it does not express (or exhibits reducedexpression) of TCR alpha, TCR beta, TCR gamma, TCR delta, TCR epsilon,and/or TCR zeta) or engineered such that it produces very littlefunctional TCR on its surface. Alternatively, the T cell can express asubstantially impaired TCR, e.g., by expression of mutated or truncatedforms of one or more of the subunits of the TCR. The term “substantiallyimpaired TCR” means that this TCR will not elicit an adverse immunereaction in a host.

A T cell described herein can be, e.g., engineered such that it does notexpress a functional HLA on its surface. For example, a T cell describedherein, can be engineered such that cell surface expression HLA, e.g.,HLA class 1 and/or HLA class II, is downregulated. In some embodiments,downregulation of HLA may be accomplished by reducing or eliminatingexpression of beta-2 microglobulin (B2M).

In some embodiments, the T cell can lack a functional TCR and afunctional HLA, e.g., HLA class I and/or HLA class II.

Modified T cells that lack expression of a functional TCR and/or HLA canbe obtained by any suitable means, including a knock out or knock downof one or more subunit of TCR or HLA. For example, the T cell caninclude a knock down of TCR and/or HLA using siRNA, shRNA, clusteredregularly interspaced short palindromic repeats (CRISPR)transcription-activator like effector nuclease (TALEN), or zinc fingerendonuclease (ZFN).

In some embodiments, the allogeneic cell can be a cell which does notexpresses or expresses at low levels an inhibitory molecule, e.g. by anymethod described herein. For example, the cell can be a cell that doesnot express or expresses at low levels an inhibitory molecule, e.g.,that can decrease the ability of a CAR-expressing cell to mount animmune effector response. Examples of inhibitory molecules include PD1,PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/orCEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86,B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHCclass I, MHC class II, GAL9, adenosine, and TGFR (e.g., TGFRbeta).Inhibition of an inhibitory molecule, e.g., by inhibition at the DNA,RNA or protein level, can optimize a CAR-expressing cell performance. Inembodiments, an inhibitory nucleic acid, e.g., an inhibitory nucleicacid, e.g., a dsRNA, e.g., an siRNA or shRNA, a clustered regularlyinterspaced short palindromic repeats (CRISPR), atranscription-activator like effector nuclease (TALEN), or a zinc fingerendonuclease (ZFN), e.g., as described herein, can be used.

siRNA and shRNA to Inhibit TCR or HLA

In some embodiments, TCR expression and/or HLA expression can beinhibited using siRNA or shRNA that targets a nucleic acid encoding aTCR and/or HLA, and/or an inhibitory molecule described herein (e.g.,PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/orCEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86,B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, WICclass I, WIC class II, GAL9, adenosine, and TGFR beta), in a cell, e.g.,T cell.

Expression systems for siRNA and shRNAs, and exemplary shRNAs, aredescribed, e.g., in paragraphs 649 and 650 of International ApplicationWO2015/142675, filed Mar. 13, 2015, which is incorporated by referencein its entirety.

CRISPR to inhibit TCR or HLA

“CRISPR” or “CRISPR to TCR and/or HLA” or “CRISPR to inhibit TCR and/orHLA” as used herein refers to a set of clustered regularly interspacedshort palindromic repeats, or a system comprising such a set of repeats.“Cas”, as used herein, refers to a CRISPR-associated protein. A“CRISPR/Cas” system refers to a system derived from CRISPR and Cas whichcan be used to silence or mutate a TCR and/or HLA gene, and/or aninhibitory molecule described herein (e.g., PD1, PD-L1, PD-L2, CTLA4,TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA,BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MEW class II,GAL9, adenosine, and TGFR beta), in a cell, e.g., T cell.

The CRISPR/Cas system, and uses thereof, are described, e.g., inparagraphs 651-658 of International Application WO2015/142675, filedMar. 13, 2015, which is incorporated by reference in its entirety.

TALEN to Inhibit TCR and/or HLA

“TALEN” or “TALEN to HLA and/or TCR” or “TALEN to inhibit HLA and/orTCR” refers to a transcription activator-like effector nuclease, anartificial nuclease which can be used to edit the HLA and/or TCR gene,and/or an inhibitory molecule described herein (e.g., PD1, PD-L1, PD-L2,CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3,VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, WIC class II,GAL9, adenosine, and TGFR beta), in a cell, e.g., T cell.

TALENs, and uses thereof, are described, e.g., in paragraphs 659-665 ofInternational Application WO2015/142675, filed Mar. 13, 2015, which isincorporated by reference in its entirety.

Zinc Finger Nuclease to Inhibit HLA and/or TCR

“ZFN” or “Zinc Finger Nuclease” or “ZFN to HLA and/or TCR” or “ZFN toinhibit HLA and/or TCR” refer to a zinc finger nuclease, an artificialnuclease which can be used to edit the HLA and/or TCR gene, and/or aninhibitory molecule described herein (e.g., PD1, PD-L1, PD-L2, CTLA4,TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA,BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, WIC class I, MHC class II,GAL9, adenosine, and TGFR beta), in a cell, e.g., T cell.

ZFNs, and uses thereof, are described, e.g., in paragraphs 666-671 ofInternational Application WO2015/142675, filed Mar. 13, 2015, which isincorporated by reference in its entirety.

Telomerase Expression

While not wishing to be bound by any particular theory, in someembodiments, a therapeutic T cell has short term persistence in apatient, due to shortened telomeres in the T cell; accordingly,transfection with a telomerase gene can lengthen the telomeres of the Tcell and improve persistence of the T cell in the patient. See CarlJune, “Adoptive T cell therapy for cancer in the clinic”, Journal ofClinical Investigation, 117:1466-1476 (2007). Thus, in an embodiment, animmune effector cell, e.g., a T cell, ectopically expresses a telomerasesubunit, e.g., the catalytic subunit of telomerase, e.g., TERT, e.g.,hTERT. In some aspects, this disclosure provides a method of producing aCAR-expressing cell, comprising contacting a cell with a nucleic acidencoding a telomerase subunit, e.g., the catalytic subunit oftelomerase, e.g., TERT, e.g., hTERT. The cell may be contacted with thenucleic acid before, simultaneous with, or after being contacted with aconstruct encoding a CAR.

In one aspect, the disclosure features a method of making a populationof immune effector cells (e.g., T cells, NK cells). In an embodiment,the method comprises: providing a population of immune effector cells(e.g., T cells or NK cells), contacting the population of immuneeffector cells with a nucleic acid encoding a CAR; and contacting thepopulation of immune effector cells with a nucleic acid encoding atelomerase subunit, e.g., hTERT, under conditions that allow for CAR andtelomerase expression.

In an embodiment, the nucleic acid encoding the telomerase subunit isDNA. In an embodiment, the nucleic acid encoding the telomerase subunitcomprises a promoter capable of driving expression of the telomerasesubunit.

In an embodiment, hTERT has the amino acid sequence of GenBank ProteinID AAC51724.1 (Meyerson et al., “hEST2, the Putative Human TelomeraseCatalytic Subunit Gene, Is Up-Regulated in Tumor Cells and duringImmortalization” Cell Volume 90, Issue 4, 22 Aug. 1997, Pages 785-795)as set out in SEQ ID NO: 82 herein.

In an embodiment, the hTERT has a sequence at least 80%, 85%, 90%, 95%,96{circumflex over ( )}, 97%, 98%, or 99% identical to the sequence ofSEQ ID NO: 82. In an embodiment, the hTERT has a sequence of SEQ ID NO:82. In an embodiment, the hTERT comprises a deletion (e.g., of no morethan 5, 10, 15, 20, or 30 amino acids) at the N-terminus, theC-terminus, or both. In an embodiment, the hTERT comprises a transgenicamino acid sequence (e.g., of no more than 5, 10, 15, 20, or 30 aminoacids) at the N-terminus, the C-terminus, or both.

In an embodiment, the hTERT is encoded by the nucleic acid sequence ofGenBank Accession No. AF018167 (Meyerson et al., “hEST2, the PutativeHuman Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cellsand during Immortalization” Cell Volume 90, Issue 4, 22 Aug. 1997, Pages785-795) as set out in SEQ ID NO: 83 herein.

In an embodiment, the hTERT is encoded by a nucleic acid having asequence at least 80%, 85%, 90%, 95%, 96, 97%, 98%, or 99% identical tothe sequence of SEQ ID NO: 83. In an embodiment, the hTERT is encoded bya nucleic acid of SEQ ID NO: 83.

Activation and Expansion of Immune Effector Cells (e.g., T Cells)

Immune effector cells such as T cells may be activated and expandedgenerally using methods as described, for example, in U.S. Pat. Nos.6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466;6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843;5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent ApplicationPublication No. 20060121005. In some embodiments, immune effector cellsare subjected to an assay as described herein (e.g., one or morebiomarkers are assayed) before, during, or after activation, or before,during, or after expansion.

Generally, a population of immune effector cells may be expanded bycontact with a surface having attached thereto an agent that stimulatesa CD3/TCR complex associated signal and a ligand that stimulates acostimulatory molecule on the surface of the T cells. In particular, Tcell populations may be stimulated as described herein, such as bycontact with an anti-CD3 antibody, or antigen-binding fragment thereof,or an anti-CD2 antibody immobilized on a surface, or by contact with aprotein kinase C activator (e.g., bryostatin) in conjunction with acalcium ionophore. For co-stimulation of an accessory molecule on thesurface of the T cells, a ligand that binds the accessory molecule isused. For example, a population of T cells can be contacted with ananti-CD3 antibody and an anti-CD28 antibody, under conditionsappropriate for stimulating proliferation of the T cells. To stimulateproliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3antibody and an anti-CD28 antibody can be used. Examples of an anti-CD28antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can beused as can other methods commonly known in the art (Berg et al.,Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med.190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63,1999).

In certain aspects, the primary stimulatory signal and the costimulatorysignal for the T cell may be provided by different protocols. Forexample, the agents providing each signal may be in solution or coupledto a surface. When coupled to a surface, the agents may be coupled tothe same surface (i.e., in “cis” formation) or to separate surfaces(i.e., in “trans” formation). Alternatively, one agent may be coupled toa surface and the other agent in solution. In one aspect, the agentproviding the costimulatory signal is bound to a cell surface and theagent providing the primary activation signal is in solution or coupledto a surface. In certain aspects, both agents can be in solution. In oneaspect, the agents may be in soluble form, and then cross-linked to asurface, such as a cell expressing Fc receptors or an antibody or otherbinding agent which will bind to the agents. In this regard, see forexample, U.S. Patent Application Publication Nos. 20040101519 and20060034810 for artificial antigen presenting cells (aAPCs) that arecontemplated for use in activating and expanding T cells in the presentinvention.

In one aspect, the two agents are immobilized on beads, either on thesame bead, i.e., “cis,” or to separate beads, i.e., “trans.” By way ofexample, the agent providing the primary activation signal is ananti-CD3 antibody or an antigen-binding fragment thereof and the agentproviding the costimulatory signal is an anti-CD28 antibody orantigen-binding fragment thereof; and both agents are co-immobilized tothe same bead in equivalent molecular amounts. In one aspect, a 1:1ratio of each antibody bound to the beads for CD4+ T cell expansion andT cell growth is used. In certain aspects of the present invention, aratio of anti CD3:CD28 antibodies bound to the beads is used such thatan increase in T cell expansion is observed as compared to the expansionobserved using a ratio of 1:1. In one particular aspect an increase offrom about 1 to about 3 fold is observed as compared to the expansionobserved using a ratio of 1:1. In one aspect, the ratio of CD3:CD28antibody bound to the beads ranges from 100:1 to 1:100 and all integervalues there between. In one aspect, more anti-CD28 antibody is bound tothe particles than anti-CD3 antibody, i.e., the ratio of CD3:CD28 isless than one. In certain aspects, the ratio of anti CD28 antibody toanti CD3 antibody bound to the beads is greater than 2:1. In oneparticular aspect, a 1:100 CD3:CD28 ratio of antibody bound to beads isused. In one aspect, a 1:75 CD3:CD28 ratio of antibody bound to beads isused. In a further aspect, a 1:50 CD3:CD28 ratio of antibody bound tobeads is used. In one aspect, a 1:30 CD3:CD28 ratio of antibody bound tobeads is used. In one preferred aspect, a 1:10 CD3:CD28 ratio ofantibody bound to beads is used. In one aspect, a 1:3 CD3:CD28 ratio ofantibody bound to the beads is used. In yet one aspect, a 3:1 CD3:CD28ratio of antibody bound to the beads is used.

Ratios of particles to cells from 1:500 to 500:1 and any integer valuesin between may be used to stimulate T cells or other target cells. Asthose of ordinary skill in the art can readily appreciate, the ratio ofparticles to cells may depend on particle size relative to the targetcell. For example, small sized beads could only bind a few cells, whilelarger beads could bind many. In certain aspects the ratio of cells toparticles ranges from 1:100 to 100:1 and any integer values in-betweenand in further aspects the ratio comprises 1:9 to 9:1 and any integervalues in between, can also be used to stimulate T cells. The ratio ofanti-CD3- and anti-CD28-coupled particles to T cells that result in Tcell stimulation can vary as noted above, however certain suitablevalues include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6,1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1,and 15:1 with one suitable ratio being at least 1:1 particles per Tcell. In one aspect, a ratio of particles to cells of 1:1 or less isused. In one particular aspect, a suitable particle: cell ratio is 1:5.In further aspects, the ratio of particles to cells can be varieddepending on the day of stimulation. For example, in one aspect, theratio of particles to cells is from 1:1 to 10:1 on the first day andadditional particles are added to the cells every day or every other daythereafter for up to 10 days, at final ratios of from 1:1 to 1:10 (basedon cell counts on the day of addition). In one particular aspect, theratio of particles to cells is 1:1 on the first day of stimulation andadjusted to 1:5 on the third and fifth days of stimulation. In oneaspect, particles are added on a daily or every other day basis to afinal ratio of 1:1 on the first day, and 1:5 on the third and fifth daysof stimulation. In one aspect, the ratio of particles to cells is 2:1 onthe first day of stimulation and adjusted to 1:10 on the third and fifthdays of stimulation. In one aspect, particles are added on a daily orevery other day basis to a final ratio of 1:1 on the first day, and 1:10on the third and fifth days of stimulation. One of skill in the art willappreciate that a variety of other ratios may be suitable for use in thepresent invention. In particular, ratios will vary depending on particlesize and on cell size and type. In one aspect, the most typical ratiosfor use are in the neighborhood of 1:1, 2:1 and 3:1 on the first day.

In further aspects, the cells, such as T cells, are combined withagent-coated beads, the beads and the cells are subsequently separated,and then the cells are cultured. In an alternative aspect, prior toculture, the agent-coated beads and cells are not separated but arecultured together. In a further aspect, the beads and cells are firstconcentrated by application of a force, such as a magnetic force,resulting in increased ligation of cell surface markers, therebyinducing cell stimulation.

By way of example, cell surface proteins may be ligated by allowingparamagnetic beads to which anti-CD3 and anti-CD28 are attached (3×28beads) to contact the T cells. In one aspect the cells (for example, 10⁴to 10⁹ T cells) and beads (for example, DYNABEADS® M-450 CD3/CD28 Tparamagnetic beads at a ratio of 1:1) are combined in a buffer, forexample PBS (without divalent cations such as, calcium and magnesium).Again, those of ordinary skill in the art can readily appreciate anycell concentration may be used. For example, the target cell may be veryrare in the sample and comprise only 0.01% of the sample or the entiresample (i.e., 100%) may comprise the target cell of interest.Accordingly, any cell number is within the context of the presentinvention. In certain aspects, it may be desirable to significantlydecrease the volume in which particles and cells are mixed together(i.e., increase the concentration of cells), to ensure maximum contactof cells and particles. For example, in one aspect, a concentration ofabout 10 billion cells/ml, 9 billion/ml, 8 billion/ml, 7 billion/ml, 6billion/ml, 5 billion/ml, or 2 billion cells/ml is used. In one aspect,greater than 100 million cells/ml is used. In a further aspect, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet one aspect, a concentration of cells from 75,80, 85, 90, 95, or 100 million cells/ml is used. In further aspects,concentrations of 125 or 150 million cells/ml can be used. Using highconcentrations can result in increased cell yield, cell activation, andcell expansion. Further, use of high cell concentrations allows moreefficient capture of cells that may weakly express target antigens ofinterest, such as CD28-negative T cells. Such populations of cells mayhave therapeutic value and would be desirable to obtain in certainaspects. For example, using high concentration of cells allows moreefficient selection of CD8+ T cells that normally have weaker CD28expression.

In one embodiment, cells transduced with a nucleic acid encoding a CAR,e.g., a CAR described herein, are expanded, e.g., by a method describedherein. In one embodiment, the cells are expanded in culture for aperiod of several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18,21 hours) to about 14 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13 or 14 days). In one embodiment, the cells are expanded for a periodof 4 to 9 days. In one embodiment, the cells are expanded for a periodof 8 days or less, e.g., 7, 6 or 5 days. In one embodiment, the cells,e.g., a CD19 CAR cell described herein, are expanded in culture for 5days, and the resulting cells are more potent than the same cellsexpanded in culture for 9 days under the same culture conditions.Potency can be defined, e.g., by various T cell functions, e.g.proliferation, target cell killing, cytokine production, activation,migration, or combinations thereof. In one embodiment, the cells, e.g.,a CD19 CAR cell described herein, expanded for 5 days show at least aone, two, three or four fold increase in cells doublings upon antigenstimulation as compared to the same cells expanded in culture for 9 daysunder the same culture conditions. In one embodiment, the cells, e.g.,the cells expressing a CD19 CAR described herein, are expanded inculture for 5 days, and the resulting cells exhibit higherproinflammatory cytokine production, e.g., IFN-γ and/or GM-CSF levels,as compared to the same cells expanded in culture for 9 days under thesame culture conditions. In one embodiment, the cells, e.g., a CD19 CARcell described herein, expanded for 5 days show at least a one, two,three, four, five, tenfold or more increase in pg/ml of proinflammatorycytokine production, e.g., IFN-γ and/or GM-CSF levels, as compared tothe same cells expanded in culture for 9 days under the same cultureconditions.

Several cycles of stimulation may also be desired such that culture timeof T cells can be 60 days or more. Conditions appropriate for T cellculture include an appropriate media (e.g., Minimal Essential Media orRPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factorsnecessary for proliferation and viability, including serum (e.g., fetalbovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4,IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α or any otheradditives for the growth of cells known to the skilled artisan. Otheradditives for the growth of cells include, but are not limited to,surfactant, plasmanate, and reducing agents such as N-acetyl-cysteineand 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM,α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added aminoacids, sodium pyruvate, and vitamins, either serum-free or supplementedwith an appropriate amount of serum (or plasma) or a defined set ofhormones, and/or an amount of cytokine(s) sufficient for the growth andexpansion of T cells. Antibiotics, e.g., penicillin and streptomycin,are included only in experimental cultures, not in cultures of cellsthat are to be infused into a subject. The target cells are maintainedunder conditions necessary to support growth, for example, anappropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5%CO₂).

In one embodiment, the cells are expanded in an appropriate media (e.g.,media described herein) that includes one or more interleukin thatresult in at least a 200-fold (e.g., 200-fold, 250-fold, 300-fold,350-fold) increase in cells over a 14 day expansion period, e.g., asmeasured by a method described herein such as flow cytometry. In oneembodiment, the cells are expanded in the presence IL-15 and/or IL-7(e.g., IL-15 and IL-7).

In embodiments, methods described herein, e.g., CAR-expressing cellmanufacturing methods, comprise removing T regulatory cells, e.g., CD25+T cells, from a cell population, e.g., using an anti-CD25 antibody, orfragment thereof, or a CD25-binding ligand, IL-2. Methods of removing Tregulatory cells, e.g., CD25+ T cells, from a cell population aredescribed herein. In embodiments, the methods, e.g., manufacturingmethods, further comprise contacting a cell population (e.g., a cellpopulation in which T regulatory cells, such as CD25+ T cells, have beendepleted; or a cell population that has previously contacted ananti-CD25 antibody, fragment thereof, or CD25-binding ligand) with IL-15and/or IL-7. For example, the cell population (e.g., that has previouslycontacted an anti-CD25 antibody, fragment thereof, or CD25-bindingligand) is expanded in the presence of IL-15 and/or IL-7.

In some embodiments a CAR-expressing cell described herein is contactedwith a composition comprising a interleukin-15 (IL-15) polypeptide, ainterleukin-15 receptor alpha (IL-15Ra) polypeptide, or a combination ofboth a IL-15 polypeptide and a IL-15Ra polypeptide e.g., hetIL-15,during the manufacturing of the CAR-expressing cell, e.g., ex vivo. Inembodiments, a CAR-expressing cell described herein is contacted with acomposition comprising a IL-15 polypeptide during the manufacturing ofthe CAR-expressing cell, e.g., ex vivo. In embodiments, a CAR-expressingcell described herein is contacted with a composition comprising acombination of both a IL-15 polypeptide and a IL-15 Ra polypeptideduring the manufacturing of the CAR-expressing cell, e.g., ex vivo. Inembodiments, a CAR-expressing cell described herein is contacted with acomposition comprising hetlL-15 during the manufacturing of theCAR-expressing cell, e.g., ex vivo.

In one embodiment the CAR-expressing cell described herein is contactedwith a composition comprising hetIL-15 during ex vivo expansion. In anembodiment, the CAR-expressing cell described herein is contacted with acomposition comprising an IL-15 polypeptide during ex vivo expansion. Inan embodiment, the CAR-expressing cell described herein is contactedwith a composition comprising both an IL-15 polypeptide and an IL-15Rapolypeptide during ex vivo expansion. In one embodiment the contactingresults in the survival and proliferation of a lymphocyte subpopulation,e.g., CD8+ T cells.

In one embodiment, the cells are cultured (e.g., expanded, simulated,and/or transduced) in media comprising serum. The serum may be, e.g.,human AB serum (hAB). In some embodiments, the hAB serum is present atabout 2%, about 5%, about 2-3%, about 3-4%, about 4-5%, or about 2-5%.As shown in Example 15 herein, 2% and 5% serum are each suitable levelsthat allow for many fold expansion of T cells. Furthermore, as shown inSmith et al., “Ex vivo expansion of human T cells for adoptiveimmunotherapy using the novel Xeno-free CTS Immune Cell SerumReplacement” Clinical & Translational Immunology (2015) 4, e31;doi:10.1038/cti.2014.31, medium containing 2% human AB serum is suitablefor ex vivo expansion of T cells.

T cells that have been exposed to varied stimulation times may exhibitdifferent characteristics. For example, typical blood or apheresedperipheral blood mononuclear cell products have a helper T cellpopulation (TH, CD4+) that is greater than the cytotoxic or suppressor Tcell population (TC, CD8+). Ex vivo expansion of T cells by stimulatingCD3 and CD28 receptors produces a population of T cells that prior toabout days 8-9 consists predominately of TH cells, while after aboutdays 8-9, the population of T cells comprises an increasingly greaterpopulation of TC cells. Accordingly, depending on the purpose oftreatment, infusing a subject with a T cell population comprisingpredominately of TH cells may be advantageous. Similarly, if anantigen-specific subset of TC cells has been isolated it may bebeneficial to expand this subset to a greater degree.

Further, in addition to CD4 and CD8 markers, other phenotypic markersvary significantly, but in large part, reproducibly during the course ofthe cell expansion process. Thus, such reproducibility enables theability to tailor an activated T cell product for specific purposes.

In some embodiments, cells transduced with a nucleic acid encoding aCAR, e.g., a CAR described herein, can be selected for administrationbased upon, e.g., protein expression levels of one or more of CCL20,GM-CSF, IFNγ, IL-10, IL-13, IL-17a, IL-2, IL-21, IL-4, IL-5, IL-6, IL-9,TNFα and/or combinations thereof. In some embodiments, cells transducedwith a nucleic acid encoding a CAR, e.g., a CAR described herein, can beselected for administration based upon, e.g., protein expression levelsof CCL20, IL-17a, IL-6 and combinations thereof.

Once a CAR described herein is constructed, various assays can be usedto evaluate the activity of the molecule, such as but not limited to,the ability to expand T cells following antigen stimulation, sustain Tcell expansion in the absence of re-stimulation, and anti-canceractivities in appropriate in vitro and animal models. Assays to evaluatethe effects of a CAR are described in further detail below.

Western blot analysis of CAR expression in primary T cells can be usedto detect the presence of monomers and dimers, e.g., as described inparagraph 695 of International Application WO2015/142675, filed Mar. 13,2015, which is herein incorporated by reference in its entirety.

In vitro expansion of CAR⁺ T cells following antigen stimulation can bemeasured by flow cytometry. For example, a mixture of CD4⁺ and CD8⁺ Tcells are stimulated with αCD3/αCD28 aAPCs followed by transduction withlentiviral vectors expressing GFP under the control of the promoters tobe analyzed. Exemplary promoters include the CMV IE gene, EF-1α,ubiquitin C, or phosphoglycerokinase (PGK) promoters. GFP fluorescenceis evaluated on day 6 of culture in the CD4⁺ and/or CD8⁺ T cell subsetsby flow cytometry. See, e.g., Milone et al., MOLECULAR THERAPY 17(8):1453-1464 (2009). Alternatively, a mixture of CD4⁺ and CD8⁺ T cells arestimulated with αCD3/αCD28 coated magnetic beads on day 0, andtransduced with CAR on day 1 using a bicistronic lentiviral vectorexpressing CAR along with eGFP using a 2A ribosomal skipping sequence.Cultures are re-stimulated with either a cancer associate antigen asdescribed herein ⁺K562 cells (K562-a cancer associate antigen asdescribed herein), wild-type K562 cells (K562 wild type) or K562 cellsexpressing hCD32 and 4-1BBL in the presence of antiCD3 and anti-CD28antibody (K562-BBL-3/28) following washing. Exogenous IL-2 is added tothe cultures every other day at 100 IU/ml. GFP⁺ T cells are enumeratedby flow cytometry using bead-based counting. See, e.g., Milone et al.,MOLECULAR THERAPY 17(8): 1453-1464 (2009).

Sustained CAR⁺ T cell expansion in the absence of re-stimulation canalso be measured. See, e.g., Milone et al., MOLECULAR THERAPY 17(8):1453-1464 (2009). Briefly, mean T cell volume (fl) is measured on day 8of culture using a Coulter Multisizer III particle counter, a NexcelomCellometer Vision or Millipore Scepter, following stimulation withαCD3/αCD28 coated magnetic beads on day 0, and transduction with theindicated CAR on day 1.

Animal models can also be used to measure a CAR-expressing cell (e.g., Tcell, NK cell) activity, e.g., as described in paragraph 698 ofInternational Application WO2015/142675, filed Mar. 13, 2015, which isherein incorporated by reference in its entirety.

Dose dependent CAR treatment response can be evaluated, e.g., asdescribed in paragraph 699 of International Application WO2015/142675,filed Mar. 13, 2015, which is herein incorporated by reference in itsentirety.

Assessment of cell proliferation and cytokine production has beenpreviously described, e.g., as described in paragraph 700 ofInternational Application WO2015/142675, filed Mar. 13, 2015, which isherein incorporated by reference in its entirety.

In another embodiment, potency of a cell (e.g., T cell, NK cell)population (e.g. a CAR-expressing cell) product, e.g., a CD19CAR-expressing cell (e.g., T cell, NK cell) cell product, e.g., CTL019cells) is assessed using a Luminex® panel of cytokines to determinecytokine expression levels. Cell (e.g., T cell, NK cell) populations(e.g, a manufactured CAR-expressing cell) cell product, e.g., a CD19CAR-expressing cell product, e.g., CTL019 cells) are activated in vitroby CD19-expressing K562 (K562-19) cells, which mimic CD19-expressing Bcells in CLL. Following cell (e.g., T cell, NK cell) activation,cytokine expression profiles are measured in the co-cultured cell mediaand potency of activated cells (e.g., a CAR-expressing cell product,e.g., a CD19 CAR-expressing cell product, e.g., CTL019 cells) iscorrelated with expression of different cytokines including, but notlimited to CCL-20/MIP-3a, GM-CSF, IFNγ, IL-10, IL-13, IL-17a, IL-2,IL-21, IL-4, IL-5, IL-6, IL-9, TNFα and/or combinations thereof.

In an embodiment, cytokine expression levels are informative withregards to the potency of a cell (e.g., T cell, NK cell) population(e.g., to kill tumor cells). In an embodiment, cytokine expressionlevels described herein are used to improve a cell (e.g., T cell, NKcell) population (e.g., a CAR-expressing cell product, e.g., a CD 19CAR-expressing cell product, e.g., CTL019 cells) prior to infusion inpatients. In an embodiment, cytokine expression levels described hereinprovide an endpoint during optimization of the manufacturing process.

Cytotoxicity can be assessed by a standard 51Cr-release assay, e.g., asdescribed in paragraph 701 of International Application WO2015/142675,filed Mar. 13, 2015, which is herein incorporated by reference in itsentirety.

Imaging technologies can be used to evaluate specific trafficking andproliferation of CARs in tumor-bearing animal models, e.g., as describedin paragraph 702 of International Application WO2015/142675, filed Mar.13, 2015, which is herein incorporated by reference in its entirety.

Other assays, including those described in the Example section herein aswell as those that are known in the art can also be used to evaluate theCARs described herein.

Alternatively, or in combination to the methods disclosed herein,methods and compositions for one or more of: detection and/orquantification of CAR-expressing cells (e.g., in vitro or in vivo (e.g.,clinical monitoring)); immune cell expansion and/or activation; and/orCAR-specific selection, that involve the use of a CAR ligand, aredisclosed. In one exemplary embodiment, the CAR ligand is an antibodythat binds to the CAR molecule, e.g., binds to the extracellular antigenbinding domain of CAR (e.g., an antibody that binds to the antigenbinding domain, e.g., an anti-idiotypic antibody; or an antibody thatbinds to a constant region of the extracellular binding domain). Inother embodiments, the CAR ligand is a CAR antigen molecule (e.g., a CARantigen molecule as described herein).

In one aspect, a method for detecting and/or quantifying CAR-expressingcells is disclosed. For example, the CAR ligand can be used to detectand/or quantify CAR-expressing cells in vitro or in vivo (e.g., clinicalmonitoring of CAR-expressing cells in a patient, or dosing a patient).The method includes:

providing the CAR ligand (optionally, a labelled CAR ligand, e.g., a CARligand that includes a tag, a bead, a radioactive or fluorescent label);

acquiring the CAR-expressing cell (e.g., acquiring a sample containingCAR-expressing cells, such as a manufacturing sample or a clinicalsample);

contacting the CAR-expressing cell with the CAR ligand under conditionswhere binding occurs, thereby detecting the level (e.g., amount) of theCAR-expressing cells present. Binding of the CAR-expressing cell withthe CAR ligand can be detected using standard techniques such as FACS,ELISA and the like.

In another aspect, a method of expanding and/or activating cells (e.g.,immune effector cells) is disclosed. The method includes:

providing a CAR-expressing cell (e.g., a first CAR-expressing cell or atransiently expressing CAR cell);

contacting said CAR-expressing cell with a CAR ligand, e.g., a CARligand as described herein), under conditions where immune cellexpansion and/or proliferation occurs, thereby producing the activatedand/or expanded cell population.

In certain embodiments, the CAR ligand is present on (e.g., isimmobilized or attached to a substrate, e.g., a non-naturally occurringsubstrate). In some embodiments, the substrate is a non-cellularsubstrate. The non-cellular substrate can be a solid support chosenfrom, e.g., a plate (e.g., a microtiter plate), a membrane (e.g., anitrocellulose membrane), a matrix, a chip or a bead. In embodiments,the CAR ligand is present in the substrate (e.g., on the substratesurface). The CAR ligand can be immobilized, attached, or associatedcovalently or non-covalently (e.g., cross-linked) to the substrate. Inone embodiment, the CAR ligand is attached (e.g., covalently attached)to a bead. In the aforesaid embodiments, the immune cell population canbe expanded in vitro or ex vivo. The method can further includeculturing the population of immune cells in the presence of the ligandof the CAR molecule, e.g., using any of the methods described herein.

In other embodiments, the method of expanding and/or activating thecells further comprises addition of a second stimulatory molecule, e.g.,CD28. For example, the CAR ligand and the second stimulatory moleculecan be immobilized to a substrate, e.g., one or more beads, therebyproviding increased cell expansion and/or activation.

In yet another aspect, a method for selecting or enriching for a CARexpressing cell is provided. The method includes contacting the CARexpressing cell with a CAR ligand as described herein; and selecting thecell on the basis of binding of the CAR ligand.

In yet other embodiments, a method for depleting, reducing and/orkilling a CAR expressing cell is provided. The method includescontacting the CAR expressing cell with a CAR ligand as describedherein; and targeting the cell on the basis of binding of the CARligand, thereby reducing the number, and/or killing, the CAR-expressingcell. In one embodiment, the CAR ligand is coupled to a toxic agent(e.g., a toxin or a cell ablative drug). In another embodiment, theanti-idiotypic antibody can cause effector cell activity, e.g., ADCC orADC activities.

Exemplary anti-CAR antibodies that can be used in the methods disclosedherein are described, e.g., in WO 2014/190273 and by Jena et al.,“Chimeric Antigen Receptor (CAR)-Specific Monoclonal Antibody to DetectCD19-Specific T cells in Clinical Trials”, PLOS March 2013 8:3 e57838,the contents of which are incorporated by reference.

In some aspects and embodiments, the compositions and methods herein areoptimized for a specific subset of T cells, e.g., as described in USSerial No. PCT/US2015/043219 filed Jul. 31, 2015, the contents of whichare incorporated herein by reference in their entirety. In someembodiments, the optimized subsets of T cells display an enhancedpersistence compared to a control T cell, e.g., a T cell of a differenttype (e.g., CD8⁺ or CD4⁺) expressing the same construct.

In some embodiments, a CD4⁺ T cell comprises a CAR described herein,which CAR comprises an intracellular signaling domain suitable for(e.g., optimized for, e.g., leading to enhanced persistence in) a CD4⁺ Tcell, e.g., an ICOS domain. In some embodiments, a CD8⁺ T cell comprisesa CAR described herein, which CAR comprises an intracellular signalingdomain suitable for (e.g., optimized for, e.g., leading to enhancedpersistence of) a CD8⁺ T cell, e.g., a 4-1BB domain, a CD28 domain, oranother costimulatory domain other than an ICOS domain. In someembodiments, the CAR described herein comprises an antigen bindingdomain described herein, e.g., a CAR comprising an antigen bindingdomain.

In an aspect, described herein is a method of treating a subject, e.g.,a subject having cancer. The method includes administering to saidsubject, an effective amount of:

-   -   1) a CD4⁺ T cell comprising a CAR (the CAR^(CD4+))

comprising:

an antigen binding domain, e.g., an antigen binding domain describedherein;

a transmembrane domain; and

an intracellular signaling domain, e.g., a first costimulatory domain,e.g., an ICOS domain; and

-   -   2) a CD8⁺ T cell comprising a CAR (the CAR^(CD8+)) comprising:

an antigen binding domain, e.g., an antigen binding domain describedherein;

a transmembrane domain; and

an intracellular signaling domain, e.g., a second costimulatory domain,e.g., a 4-1BB domain, a CD28 domain, or another costimulatory domainother than an ICOS domain;

wherein the CAR^(CD4+) and the CAR^(CD8+) differ from one another.

Optionally, the method further includes administering:

-   -   3) a second CD8+ T cell comprising a CAR (the second CAR^(CD8+))        comprising:

an antigen binding domain, e.g., an antigen binding domain describedherein;

a transmembrane domain; and

an intracellular signaling domain, wherein the second CAR^(CD8+)comprises an intracellular signaling domain, e.g., a costimulatorysignaling domain, not present on the CAR^(CD8+), and, optionally, doesnot comprise an ICOS signaling domain.

RNA Transfection

Disclosed herein are methods for producing an in vitro transcribed RNACAR. RNA CAR and methods of using the same are described, e.g., inparagraphs 553-570 of in International Application WO2015/142675, filedMar. 13, 2015, which is herein incorporated by reference in itsentirety.

In one embodiment, the in vitro transcribed RNA CAR can be introduced toa cell as a form of transient transfection. The RNA may have a 3′ UTR, a5′ UTR, or both. The 5′ UTR may contain a Kozak sequence. The RNA maycomprise an IRES. The RNA may comprise a 5′ cap. The RNA may comprise apolyA sequence. RNA can be produced using a DNA template that comprisesa promoter, e.g., a T7, T7, or SP6 promoter. RNA can be introduced intotarget cells using any of a number of different methods, for instance,commercially available methods which include, but are not limited to,electroporation, the Gene Pulser II, Multiporator, cationic liposomemediated transfection using lipofection, polymer encapsulation, peptidemediated transfection, or biolistic particle delivery systems such as“gene guns”.

Non-Viral Delivery Methods

In some aspects, non-viral methods can be used to deliver a nucleic acidencoding a CAR described herein into a cell or tissue or a subject.Suitable non-viral delivery methods include transposons (e.g., SleepingBeauty, piggyBac, and pT2-based transposons). Exemplary non-viraldelivery methods and methods of using the same are described, e.g., inparagraphs 571-579 of International Application WO2015/142675, filedMar. 13, 2015, which is herein incorporated by reference in itsentirety.

Methods of Manufacture/Production

In one aspect, methods of manufacturing a CAR-expressing cell accordingto the invention are disclosed herein (e.g., in “Source of Cells” and“Activation and Expansion of Cells”).

In an embodiment, a method of manufacturing a CAR-expressing cell isprovided. The method comprises:

-   -   providing a preparation of a CAR-expressing cell (e.g., a        plurality of CAR-expressing immune effector cells, such as a T        cells, or an NK cells) (e.g., a CD19 CAR-expressing cell as        described herein, such as, e.g., CTL019);    -   acquiring a value for the level of (e.g., determining the level        of expression of) one or more genes listed in Table 1A, Table        1B, Table 7A, Table 7B, Table 8, Table 9, Table 10, Table 14,        Table 15, Table 16 (e.g., CCL20, IL-17a and/or IL-6), Table 17,        Table 18, Table 20, PD-1, LAG-3, TIM-3, CD57, CD27, CD122,        CD62L, and KLRG1 to obtain a gene expression pattern for the        sample;    -   (optionally) comparing the obtained gene expression pattern to        that of a historical record of gene expression;

determining a difference between the obtained and historical geneexpression; and

-   -   recording the determined difference in a quality control record.

In an embodiment, provided methods comprise steps of providing aCAR-expressing cell (e.g., T cell, NK cell) preparation (e.g., a CD19CAR-expressing cell (e.g., T cell, NK cell) as described herein, suchas, e.g., CTL019);

-   -   determining the levels of expression of one or more genes listed        in Table 1A, Table 1B, Table 7A, Table 7B, Table 8, Table 9,        Table 10, Table 14, Table 15, Table 16 (e.g., CCL20, IL-17a        and/or IL-6), Table 17, Table 18, Table 20, PD-1, LAG-3, TIM-3,        CD57, CD27, CD122, CD62L, and KLRG1 to obtain a gene expression        pattern (e.g., a gene signature) for the sample;    -   correlating the gene signature with patient response to a        CAR-expressing cell (e.g., T cell, NK cell) therapy (e.g. a CD19        CAR-expressing cell (e.g., T cell, NK cell) as described herein,        such as, e.g., CTL019);    -   and optimizing the CAR-expressing cell (e.g., T cell, NK cell)        preparation based on the correlation of the gene signature and        patient response prior to infusion into patients.

In an embodiment, provided methods comprise acquiring a value for thelevel (e.g., determining the expression level) of a cytokine, e.g., oneor more cytokines listed in Table 14, Table 15, Table 16 (e.g., CCL20,IL-17a and/or IL-6), and Table 17, Table 18, Table 20, secreted byCAR-expressing cells (e.g., T cell, NK cell) in response to antigenrecognition. In an embodiment, provided methods comprise determining theexpression levels of one or more cytokines CCL20/MIP3a, IL-17a, IL-6and/or combinations thereof, secreted by CAR-expressing cells (e.g., Tcell, NK cell) in response to antigen recognition. In an embodiment,provided methods further comprise integration of cytokines secreted byCAR-expressing cells (e.g., T cells, NK cells), e.g., one or morecytokines listed in Table 14, Table 15 and Table 16, in a potency assay.In an embodiment, provided methods further comprise integration ofcytokines CCL20/MIP3a, IL-17a, IL-6 and/or combinations thereof, in apotency assay.

In an embodiment, provided methods comprise integration of cytokinessecreted by CAR-expressing cells (e.g., T cells, NK cells), e.g., one ormore cytokines listed in Table 14, Table 15, Table 16 (e.g., CCL20,IL-17a and/or IL-6), and Table 17 in a potency assay, and determiningwhether a CAR-expressing cell (e.g., T cell, NK cell) preparation (e.g.,a CD19 CAR-expressing cell (e.g., T cell, NK cell) as described herein,such as, e.g., CTL019) may have a clinical effect. In an embodiment,CCL20/MIP3a, IL-17a, IL-6 and/or combinations thereof are used in apotency assay to determine whether a CAR-expressing cell (e.g., T cell,NK cell) preparation (e.g., a CD19 CAR-expressing cell (e.g., T cell, NKcell) as described herein, such as, e.g., CTL019) may have a clinicaleffect. In an embodiment, provided methods further comprise adjustingthe CAR-expressing cell (e.g., T cell, NK cell) infusion dose to achieveclinical efficacy.

In an embodiment, provided methods comprise a step of providing a bloodsample, e.g., a T cell sample, from a subject having cancer.

In an embodiment, provided methods further comprise a step of comparingthe obtained gene expression pattern difference with that of a referencesample.

In an embodiment, a reference sample is a CAR-expressing cell (e.g., Tcell, NK cell) preparation (e.g., a CD19 CAR-expressing cell asdescribed herein, such as, e.g., CTL019) from a different batch of cellsproducing the therapeutic CAR-expressing cell preparation.

In an embodiment, a reference sample is a healthy donor sample with amanufactured CAR-expressing cell (e.g., T cell, NK cell) product (e.g.,a CD19 CAR-expressing cell as described herein, such as, e.g., CTL019).In an embodiment, a reference sample is a healthy donor sample with amanufactured CD19 CAR-expressing cell product, such as, e.g., CTL019product.

In an embodiment, provided methods further comprise a step of recordingthe result of the comparing in a quality control record for thetherapeutic CAR-expressing cell (e.g., T cell, NK cell) preparation.

In an embodiment, the determined difference is compared with ahistorical record of the reference sample.

In an embodiment, the CAR-expressing cell (e.g., T cell, NK cell)preparation is a CD19 CAR-expressing cell (e.g., CTL019) preparation.

In an embodiment, the CAR-expressing cell (e.g., T cell, NK cell)preparation comprises a CD19 CAR-expressing cell (e.g., CTL019)preparation.

In an embodiment, the CAR-expressing cell (e.g., T cell, NK cell)preparation consists of a CD19 CAR-expressing cell (e.g., CTL019)preparation.

In an aspect, a method is provided, comprising:

providing a blood sample, e.g., a T cell sample, from a subject havingcancer;

determining the levels of expression of one or more genes listed inTable 1A, Table 1B, Table 7A, Table 7B, Table 8, Table 9, Table 10,Table 14 (e.g., CCL20, IL-17a and/or IL-6), Table 17, Table 18, Table20, PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L, or KLRG1 to obtain agene expression pattern for the sample;

comparing the obtained gene expression pattern to that of a referencevalue, e.g., a historical record of gene expression;

determining a difference between the obtained and the reference value;and

recording the determined difference in a quality control record.

The method can comprise a step of comparing the obtained gene expressionpattern difference with that of a reference sample.

In some embodiments, the methods disclosed herein further includeadministering a T cell depleting agent after treatment with the cell(e.g., an immune effector cell as described herein), thereby reducing(e.g., depleting) the CAR-expressing cells (e.g., the CD19CAR-expressingcells). Such T cell depleting agents can be used to effectively depleteCAR-expressing cells (e.g., CD19CAR-expressing cells) to mitigatetoxicity. In some embodiments, the CAR-expressing cells weremanufactured according to a method herein, e.g., assayed (e.g., beforeor after transfection or transduction) according to a method herein.

In some embodiments, the T cell depleting agent is administered one,two, three, four, or five weeks after administration of the cell, e.g.,the population of immune effector cells, described herein.

In one embodiment, the T cell depleting agent is an agent that depletesCAR-expressing cells, e.g., by inducing antibody dependent cell-mediatedcytotoxicity (ADCC) and/or complement-induced cell death. For example,CAR-expressing cells described herein may also express an antigen (e.g.,a target antigen) that is recognized by molecules capable of inducingcell death, e.g., ADCC or complement-induced cell death. For example,CAR expressing cells described herein may also express a target protein(e.g., a receptor) capable of being targeted by an antibody or antibodyfragment. Examples of such target proteins include, but are not limitedto, EpCAM, VEGFR, integrins (e.g., integrins αvβ3, α4, αI3/4β3, α4β7,α5β1, αvβ3, αv), members of the TNF receptor superfamily (e.g.,TRAIL-R1, TRAIL-R2), PDGF Receptor, interferon receptor, folatereceptor, GPNMB, ICAM-1, HLA-DR, CEA, CA-125, MUC1, TAG-72, IL-6receptor, 5T4, GD2, GD3, CD2, CD3, CD4, CD5, CD11, CD11a/LFA-1, CD15,CD18/ITGB2, CD19, CD20, CD22, CD23/1gE Receptor, CD25, CD28, CD30, CD33,CD38, CD40, CD41, CD44, CD51, CD52, CD62L, CD74, CD80, CD125,CD147/basigin, CD152/CTLA-4, CD154/CD40L, CD195/CCR5, CD319/SLAMF7, andEGFR, and truncated versions thereof (e.g., versions preserving one ormore extracellular epitopes but lacking one or more regions within thecytoplasmic domain).

In some embodiments, the CAR expressing cell co-expresses the CAR andthe target protein, e.g., naturally expresses the target protein or isengineered to express the target protein. For example, the cell, e.g.,the population of immune effector cells, can include a nucleic acid(e.g., vector) comprising the CAR nucleic acid (e.g., a CAR nucleic acidas described herein) and a nucleic acid encoding the target protein.

In one embodiment, the T cell depleting agent is a CD52 inhibitor, e.g.,an anti-CD52 antibody molecule, e.g., alemtuzumab.

In other embodiments, the cell, e.g., the population of immune effectorcells, expresses a CAR molecule as described herein (e.g., CD19CAR) andthe target protein recognized by the T cell depleting agent. In oneembodiment, the target protein is CD20. In embodiments where the targetprotein is CD20, the T cell depleting agent is an anti-CD20 antibody,e.g., rituximab.

In further embodiments of any of the aforesaid methods, the methodsfurther include transplanting a cell, e.g., a hematopoietic stem cell,or a bone marrow, into the mammal.

In another aspect, the invention features a method of conditioning amammal prior to cell transplantation. The method includes administeringto the mammal an effective amount of the cell comprising a CAR nucleicacid or polypeptide, e.g., a CD19 CAR nucleic acid or polypeptide. Insome embodiments, the cell transplantation is a stem celltransplantation, e.g., a hematopoietic stem cell transplantation, or abone marrow transplantation. In other embodiments, conditioning asubject prior to cell transplantation includes reducing the number oftarget-expressing cells in a subject, e.g., CD19-expressing normal cellsor CD19-expressing cancer cells.

Nucleic Acid Constructs Encoding a CAR

Nucleic acid molecules encoding one or more CAR constructs can beintroduced into an immune effector cell (e.g., a T cell) as describedherein. In one aspect, the nucleic acid molecule is provided as amessenger RNA transcript. In one aspect, the nucleic acid molecule isprovided as a DNA construct.

In some embodiments, a nucleic acid described herein is introduced intoa cell that has been assayed by a method described herein, e.g., one ormore biomarkers has been assayed. In some embodiments, a cell comprisinga nucleic acid described herein is assayed by a method described herein,e.g., one or more biomarkers has been assayed.

The nucleic acid molecules described herein can be a DNA molecule, anRNA molecule, or a combination thereof. In one embodiment, the nucleicacid molecule is an mRNA encoding a CAR polypeptide as described herein.In other embodiments, the nucleic acid molecule is a vector thatincludes any of the aforesaid nucleic acid molecules.

Nucleic acid molecules can encode, e.g., a CAR molecule describedherein, and can comprise, e.g., a nucleic acid sequence describedherein, e.g., in Table 11, Table 12 or Table 13.

The nucleic acid sequences coding for the desired molecules can beobtained using recombinant methods known in the art, such as, forexample by screening libraries from cells expressing the gene, byderiving the gene from a vector known to include the same, or byisolating directly from cells and tissues containing the same, usingstandard techniques. Alternatively, the gene of interest can be producedsynthetically, rather than cloned.

Also described are vectors in which a nucleic acid of the presentdisclosure is inserted. Vectors derived from retroviruses such as thelentivirus are suitable tools to achieve long-term gene transfer sincethey allow long-term, stable integration of a transgene and itspropagation in daughter cells. Lentiviral vectors have the addedadvantage over vectors derived from onco-retroviruses such as murineleukemia viruses in that they can transduce non-proliferating cells,such as hepatocytes. They also have the added advantage of lowimmunogenicity. A retroviral vector may also be, e.g., a gammaretroviralvector. A gammaretroviral vector may include, e.g., a promoter, apackaging signal (w), a primer binding site (PBS), one or more (e.g.,two) long terminal repeats (LTR), and a transgene of interest, e.g., agene encoding a CAR. A gammaretroviral vector may lack viral structuralgens such as gag, pol, and env. Exemplary gammaretroviral vectorsinclude Murine Leukemia Virus (MLV), Spleen-Focus Forming Virus (SFFV),and Myeloproliferative Sarcoma Virus (MPSV), and vectors derivedtherefrom. Other gammaretroviral vectors are described, e.g., in TobiasMaetzig et al., “Gammaretroviral Vectors: Biology, Technology andApplication” Viruses. 2011 June; 3(6): 677-713.

In another embodiment, the vector comprising the nucleic acid encodingthe desired CAR of the invention is an adenoviral vector (A5/35). Inanother embodiment, the expression of nucleic acids encoding CARs can beaccomplished using of transposons such as sleeping beauty, CRISPR, CAS9,and zinc finger nucleases. See below June et al. 2009 NATURE REVIEWSIMMUNOLOGY 9.10: 704-716, is incorporated herein by reference.

In brief summary, the expression of natural or synthetic nucleic acidsencoding CARs is typically achieved by operably linking a nucleic acidencoding the CAR polypeptide or portions thereof to a promoter, andincorporating the construct into an expression vector. The vectors canbe suitable for replication and integration eukaryotes. Typical cloningvectors contain transcription and translation terminators, initiationsequences, and promoters useful for regulation of the expression of thedesired nucleic acid sequence.

The expression constructs may also be used for nucleic acid immunizationand gene therapy, using standard gene delivery protocols. Methods forgene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346,5,580,859, 5,589,466, incorporated by reference herein in theirentireties. In another embodiment, the invention provides a gene therapyvector.

The nucleic acid can be cloned into a number of types of vectors. Forexample, the nucleic acid can be cloned into a vector including, but notlimited to a plasmid, a phagemid, a phage derivative, an animal virus,and a cosmid. Vectors of particular interest include expression vectors,replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al., 2012, MOLECULAR CLONING: ALABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers, (e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, adenovirus vectorsare used. A number of adenovirus vectors are known in the art. In oneembodiment, lentivirus vectors are used.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave been shown to contain functional elements downstream of the startsite as well. The spacing between promoter elements frequently isflexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription. Exemplary promoters include theCMV IE gene, EF-1α, ubiquitin C, or phosphoglycerokinase (PGK)promoters.

An example of a promoter that is capable of expressing a CAR transgenein a mammalian T cell is the EF1a promoter. The native EF1a promoterdrives expression of the alpha subunit of the elongation factor-1complex, which is responsible for the enzymatic delivery of aminoacyltRNAs to the ribosome. The EF1a promoter has been extensively used inmammalian expression plasmids and has been shown to be effective indriving CAR expression from transgenes cloned into a lentiviral vector.See, e.g., Milone et al., MOL. THER. 17(8): 1453-1464 (2009). In oneaspect, the EF1a promoter comprises the sequence provided as SEQ IDNO:11.

Another example of a promoter is the immediate early cytomegalovirus(CMV) promoter sequence. This promoter sequence is a strong constitutivepromoter sequence capable of driving high levels of expression of anypolynucleotide sequence operatively linked thereto. However, otherconstitutive promoter sequences may also be used, including, but notlimited to the simian virus 40 (SV40) early promoter, mouse mammarytumor virus (MMTV), human immunodeficiency virus (HIV) long terminalrepeat (LTR) promoter, MoMuLV promoter, an avian leukemia viruspromoter, an Epstein-Barr virus immediate early promoter, a Rous sarcomavirus promoter, as well as human gene promoters such as, but not limitedto, the actin promoter, the myosin promoter, the elongation factor-1apromoter, the hemoglobin promoter, and the creatine kinase promoter.Further, the invention should not be limited to the use of constitutivepromoters. Inducible promoters are also contemplated as part of theinvention. The use of an inducible promoter provides a molecular switchcapable of turning on expression of the polynucleotide sequence which itis operatively linked when such expression is desired, or turning offthe expression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

Another example of a promoter is the phosphoglycerate kinase (PGK)promoter. In embodiments, a truncated PGK promoter (e.g., a PGK promoterwith one or more, e.g., 1, 2, 5, 10, 100, 200, 300, or 400, nucleotidedeletions when compared to the wild-type PGK promoter sequence) may bedesired.

A vector may also include, e.g., a signal sequence to facilitatesecretion, a polyadenylation signal and transcription terminator (e.g.,from Bovine Growth Hormone (BGH) gene), an element allowing episomalreplication and replication in prokaryotes (e.g. SV40 origin and ColE1or others known in the art) and/or elements to allow selection (e.g.,ampicillin resistance gene and/or zeocin marker).

In order to assess the expression of a CAR polypeptide or portionsthereof, the expression vector to be introduced into a cell can alsocontain either a selectable marker gene or a reporter gene or both tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In other aspects, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers include, for example, antibiotic-resistance genes,such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Reportergenes are described, e.g., in paragraph 599 of International ApplicationWO2015/142675, filed Mar. 13, 2015, which is herein incorporated byreference in its entirety.

In embodiments, the vector may comprise two or more nucleic acidsequences encoding a CAR, e.g., a CAR described herein, e.g., a CD19CAR, and a second CAR, e.g., an inhibitory CAR or a CAR thatspecifically binds to an antigen other than CD19. In such embodiments,the two or more nucleic acid sequences encoding the CAR are encoded by asingle nucleic molecule in the same frame and as a single polypeptidechain. In this aspect, the two or more CARs, can, e.g., be separated byone or more peptide cleavage sites. (e.g., an auto-cleavage site or asubstrate for an intracellular protease). Examples of peptide cleavagesites include T2A, P2A, E2A, or F2A sites.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans, e.g., those described in paragraphs 601-603 of InternationalApplication WO2015/142675, filed Mar. 13, 2015, which is hereinincorporated by reference in its entirety.

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo), and is described, e.g., in paragraphs604-605 of International Application WO2015/142675, filed Mar. 13, 2015,which is herein incorporated by reference in its entirety.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentinvention, in order to confirm the presence of the recombinant DNAsequence in the host cell, a variety of assays may be performed. Suchassays include, for example, “molecular biological” assays well known tothose of skill in the art, such as Southern and Northern blotting,RT-PCR and PCR; “biochemical” assays, such as detecting the presence orabsence of a particular peptide, e.g., by immunological means (ELISAsand Western blots) or by assays described herein to identify agentsfalling within the scope of the invention.

Therapeutic Methods

In one aspect, the disclosure provides methods for treating a diseaseassociated with expression of a tumor antigen described herein. In someembodiments, immune effector cells are assayed by a method describedherein, e.g., one or more biomarkers is assayed, and the cells areadministered to a subject as part of a treatment described herein. Forexample, the immune effector cells can be administered as part of acombination therapy described herein.

In one aspect, the present disclosure provides methods of treatingcancer (e.g., a hematological cancer such as ALL and CLL) by providingto the subject in need thereof immune effector cells (e.g., T cells, NKcells) that are engineered to express a CAR. In one embodiment, thecancer to be treated is a B cell malignancy. In one embodiment, thecancer to be treated is ALL (acute lymphoblastic leukemia), CLL (chroniclymphocytic leukemia), DLBCL (diffuse large B-cell lymphoma), MCL(Mantle cell lymphoma, or MM (multiple myeloma).

In one aspect, the disclosure provides methods of treating cancer (e.g.,a hematological cancer such as ALL and CLL) by providing to the subjectin need thereof immune effector cells (e.g., T cells, NK cells) that areengineered to express a CD19 CAR, wherein the cancer cells express CD19.In one embodiment, the cancer to be treated is a B cell malignancy. Inone embodiment, the cancer to be treated is ALL (acute lymphoblasticleukemia), CLL (chronic lymphocytic leukemia), DLBCL (diffuse largeB-cell lymphoma), MCL (Mantle cell lymphoma), Hodgkin lymphoma, or MM(multiple myeloma).

In one aspect, the present invention provides methods of treating cancer(e.g., a hematological cancer such as ALL and CLL) by providing to thesubject in need thereof immune effector cells (e.g., T cells, NK cells)that are engineered to express a CD22 CAR, wherein the cancer cellsexpress CD22. In one embodiment, the cancer to be treated is a B cellmalignancy. In one embodiment, the cancer to be treated is ALL (acutelymphoblastic leukemia), CLL (chronic lymphocytic leukemia), DLBCL(diffuse large B-cell lymphoma), MCL (Mantle cell lymphoma), Hodgkinlymphoma, or MM (multiple myeloma).

In one aspect, the present invention provides methods of treating cancer(e.g., a hematological cancer such as ALL and CLL) by providing to thesubject in need thereof immune effector cells (e.g., T cells, NK cells)that are engineered to express a CD20 CAR, wherein the cancer cellsexpress CD20. In one embodiment, the cancer to be treated is a B cellmalignancy. In one embodiment, the cancer to be treated is ALL (acutelymphoblastic leukemia), CLL (chronic lymphocytic leukemia), DLBCL(diffuse large B-cell lymphoma), MCL (Mantle cell lymphoma), Hodgkinlymphoma, or MM (multiple myeloma).

In one aspect, the present invention provides methods of treating cancer(e.g., a hematological cancer such as ALL and CLL) by providing to thesubject in need thereof immune effector cells (e.g., T cells, NK cells)that are engineered to express a ROR1 CAR, wherein the cancer cellsexpress ROR1. In one embodiment, the cancer to be treated is a B cellmalignancy. In one embodiment, the cancer to be treated is ALL (acutelymphoblastic leukemia), CLL (chronic lymphocytic leukemia), DLBCL(diffuse large B-cell lymphoma), MCL (Mantle cell lymphoma), Hodgkinlymphoma, or MM (multiple myeloma).

The disclosure includes a type of cellular therapy where immune effectorcells (e.g., T cells, NK cells) are genetically modified (e.g., viatransduction of a lentiviral vector) to express a CAR and theCAR-expressing cell is infused to a recipient in need thereof. Theinfused cell is able to kill tumor cells in the recipient. Unlikeantibody therapies, CAR-modified immune effector cells (e.g., T cells,NK cells) are able to replicate in vivo resulting in long-termpersistence that can lead to sustained tumor control. CAR-expressingcells (e.g., T cells or NK cells) generated using lentiviral vectorswill have stable CAR expression. In various aspects, the immune effectorcells (e.g., T cells, NK cells) administered to the patient, or theirprogeny, persist in the patient for at least four months, five months,six months, seven months, eight months, nine months, ten months, elevenmonths, twelve months, thirteen months, fourteen month, fifteen months,sixteen months, seventeen months, eighteen months, nineteen months,twenty months, twenty-one months, twenty-two months, twenty-threemonths, two years, three years, four years, or five years afteradministration of the T cell to the patient.

The invention also includes a type of cellular therapy where immuneeffector cells (e.g., T cells, NK cells) are modified, e.g., by in vitrotranscribed RNA, to transiently express a CAR and the CAR-expressingcell is infused to a recipient in need thereof. CAR-expressing cells(e.g., T cells, NK cells) generated through transduction of CAR RNA(e.g., by transfection or electroporation) transiently express RNA CARsfor 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 days after transduction.The infused cell is able to kill tumor cells in the recipient. Thus, invarious aspects, the immune effector cells (e.g., T cells, NK cells)administered to the patient, is present for less than one month, e.g.,three weeks, two weeks, one week, after administration of the T cell tothe patient.

In one aspect, the present disclosure provides methods of treatingcancer (e.g., a hematological cancer such as ALL and CLL) by providingto the subject in need thereof immune effector cells (e.g., T cells, NKcells) that are engineered to express a CAR, e.g., a CAR describedherein.

In one embodiment, the present disclosure provides methods of treatingcancer (e.g., a hematological cancer such as ALL and CLL) by providingto the subject in need thereof immune effector cells (e.g., T cells, NKcells) that are engineered to express a CAR that specifically targets orbinds to a tumor antigen (or cancer associated antigen) describedherein, wherein the subject has been identified as a responder orpartial responder. In other embodiments, the methods provide treating acancer (e.g., a hematological cancer such as ALL and CLL) as a partialresponder or non-responder by providing to the subject a cancer therapyother than a CAR therapy, e.g., providing the subject a treatment thatis the standard of care for that particular type of cancer. In yetanother embodiment, the method of treatment includes altering themanufacturing of a CAR-expressing cell to enrich for naïve T cells,e.g., as described herein, for a subject identified as a partialresponder or non-responder prior to administering a CAR-expressing cell,e.g., a CAR-expressing cell described herein.

In one embodiment, the immune effector cells (e.g., T cells, NK cells)are engineered to express CD19 CAR, for treating a subject having cancer(e.g., a hematological cancer such as ALL and CLL), wherein the cancercells express CD19. In one embodiment, the cancer to be treated is ALLor CLL. The CD19 CAR molecules to be expressed in an immune effectorcell can comprise any anti-CD19 antigen binding domain in the art (e.g.,those provided in Table 12) in combination with any of the CAR domainsdescribed herein to generate a full CAR construct. For example, the fullCAR construct is a CAR listed in Table 13. Table 13 provides theexemplary full CD19 CAR constructs generated using the various CARdomains (e.g., transmembrane and intracellular signaling domains) listedin Table 12, and the anti-CD19 antigen binding domains listed in Table12. Amino acid sequences are designated (aa) and nucleic acid sequencesare designated (nt).

TABLE 13 CD19 CAR Constructs Name Sequence CAR 1 104875atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccga CAR 1-aattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccctgtcttFull-ntgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactactcttcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg (SEQ ID NO: 55) 104875MALPVTALLLPLALLLHAARPeivmtqspatlslspgeratlsc rasgdiskyln wyqqkpgqapCAR 1- rlliy htsrlhs giparfsgsgsgtdytltisslqpedfavyfc qqgntlpytfgqgtkleikgg Full-aa ggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslp dygvswirqppgkglewig viw gsettyyssslks rvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdy wgqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrykfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmgalppr (SEQ ID NO: 56) CAR 2 104876atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccga CAR 2-aattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccctgtcttFull-ntgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactaccaatcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg (SEQ ID NO: 57) 104876MALPVTALLLPLALLLHAARPeivmtqspatlslspgeratlsc rasqdiskyln wyqqkpgqapCAR 2- rlliy htsrlhs giparfsgsgsgtdytltisslqpedfavyfc qqgntlpytfgqgtkleikgg Full-aa ggsggggsggggsqvglqesgpglvkpsetlsltctvsgvslp dygvswirqppgkglewig viw gsettyyqsslks rvtiskdnsknqvslklssvtaadtavyycakhyyyggsy amdywgqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvgttgeedgcscrfpeeeeggcelrykfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (SEQ ID NO: 58) CAR 3 104877atggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgctcgcccaca CAR 3-agtccagcttcaagaatcagggcctggtctggtgaagccatctgagactctgtccctcacttgcaFull-ntccgtgagcggagtgtccctcccagactacggagtgagctggattagacagcctcccggaaagggactggagtggatcggagtgatttggggtagcgaaaccacttactattcatcttccctgaagtcacgggtcaccatttcaaaggataactcaaagaatcaagtgagcctcaagctctcatcagtcaccgccgctgacaccgccgtgtattactgtgccaagcattactactatggagggtcctacgccatggactactggggccagggaactctggtcactgtgtcatctggtggaggaggtagcggaggaggcgggagcggtggaggtggctccgaaatcgtgatgacccagagccctgcaaccctgtccctttctcccggggaacgggctaccctttcttgtcgggcatcacaagatatctcaaaatacctcaattggtatcaacagaagccgggacaggcccctaggcttcttatctaccacacctctcgcctgcatagcgggattcccgcacgctttagcgggtctggaagcgggaccgactacactctgaccatctcatctctccagcccgaggacttcgccgtctacttctgccagcagggtaacaccctgccgtacaccttcggccagggcaccaagcttgagatcaaaaccactactcccgctccaaggccacccacccctgccccgaccatcgcctctcagccgctttccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg (SEQ ID NO: 59) 104877MALPVTALLLPLALLLHAARPqvqlqesgpglvkpsetlsltctvsgvslp dygvs wirqppgkgCAR 3- lewig viwgsettyyssslks rvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdy Full-aa wgggtivtvssggggsggggsggggseivmtgspatls1spgeratlscrasqdiskyln wyggk pgqaprlliy htsrlhs giparfsgsgsgtdytltisslqpedfavyfcqqgntlpyt fgqgtkleiktttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkgpfmrpvqttqeedgcscrfpeeeeggcelrykfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (SEQ ID NO: 60) CAR 4 104878atggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgctcgcccaca CAR 4-agtccagcttcaagaatcagggcctggtctggtgaagccatctgagactctgtccctcacttgcaFull-ntccgtgagcggagtgtccctcccagactacggagtgagctggattagacagcctcccggaaagggactggagtggatcggagtgatttggggtagcgaaaccacttactatcaatcttccctgaagtcacgggtcaccatttcaaaggataactcaaagaatcaagtgagcctcaagctctcatcagtcaccgccgctgacaccgccgtgtattactgtgccaagcattactactatggagggtcctacgccatggactactggggccagggaactctggtcactgtgtcatctggtggaggaggtagcggaggaggcgggagcggtggaggtggctccgaaatcgtgatgacccagagccctgcaaccctgtccctttctcccggggaacgggctaccctttcttgtcgggcatcacaagatatctcaaaatacctcaattggtatcaacagaagccgggacaggcccctaggcttcttatctaccacacctctcgcctgcatagcgggattcccgcacgctttagcgggtctggaagcgggaccgactacactctgaccatctcatctctccagcccgaggacttcgccgtctacttctgccagcagggtaacaccctgccgtacaccttcggccagggcaccaagcttgagatcaaaaccactactcccgctccaaggccacccacccctgccccgaccatcgcctctcagccgctttccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg (SEQ ID NO: 61) 104878MALPVTALLLPLALLLHAARPqvqlqesgpglvkpsetlsltctvsgvslp dygys wirqppgkgCAR 4- lewig viwgsettyyqsslks rvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdy Full-aa wgqgtlvtvssggggsggggsggggseivmtgspatlslspgeratlscrasqdiskyln wyqqk pgqaprlliy htsrlhs giparfsgsgsgtdytltisslqpedfavyfcqqgntlpyt fgqgtkleiktttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglygglstatkdtydalhmqalppr (SEQ ID NO: 62) CAR 5 CAR5 scFveivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgs domaingsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygyswirqppgkglewigviwgsettyyssslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss (SEQ ID NO: 63)104879 atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgaCAR 5- aattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccctgtcttFull-ntgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagcggcggaggcgggagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactactcttcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg (SEQ ID NO: 64) 104879MALPVTALLLPLALLLHAARPeivmtqspatlslspgerat1sc rasgdiskyln wyqqkpgqapCAR 5- rlliy htsrlhs giparfsgsgsgtdytltisslqpedfavyfc qqgntlpytfgqgtkleikgg Full-aa ggsggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvs wirqppgkglew ig viwgsettyyssslks rvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdy wgqgtivtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkgpfmrpvqttqeedgcscrfpeeeeggcelrykfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmgalppr (SEQ ID NO: 65) CAR 6 104880atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccga CAR6-aattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccctgtcttFull-ntgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagcggaggcggagggagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactaccaatcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg (SEQ ID NO: 66) 104880MALPVTALLLPLALLLHAARPeivmtqspatlslspgeratlsc rasqdiskyln wyqqkpgqapCAR6- rlliy htsrlhs giparfsgsgsgtdytltisslqpedfavyfc qqgntlpytfgqgtkleikgg Full-aa ggsggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvs wirqppgkglew ig viwgsettyyqsslks rvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdy wgqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvgttgeedgcscrfpeeeeggcelrykfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelgkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (SEQ ID NO: 67) CAR 7 104881atggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgctcgcccaca CAR 7agtccagcttcaagaatcagggcctggtctggtgaagccatctgagactctgtccctcacttgcaFull-ntccgtgagcggagtgtccctcccagactacggagtgagctggattagacagcctcccggaaagggactggagtggatcggagtgatttggggtagcgaaaccacttactattcatcttccctgaagtcacgggtcaccatttcaaaggataactcaaagaatcaagtgagcctcaagctctcatcagtcaccgccgctgacaccgccgtgtattactgtgccaagcattactactatggagggtcctacgccatggactactggggccagggaactctggtcactgtgtcatctggtggaggaggtagcggaggaggcgggagcggtggaggtggctccggaggtggcggaagcgaaatcgtgatgacccagagccctgcaaccctgtccctttctcccggggaacgggctaccctttcttgtcgggcatcacaagatatctcaaaatacctcaattggtatcaacagaagccgggacaggcccctaggcttcttatctaccacacctctcgcctgcatagcgggattcccgcacgctttagcgggtctggaagcgggaccgactacactctgaccatctcatctctccagcccgaggacttcgccgtctacttctgccagcagggtaacaccctgccgtacaccttcggccagggcaccaagcttgagatcaaaaccactactcccgctccaaggccacccacccctgccccgaccatcgcctctcagccgctttccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg (SEQ ID NO: 68) 104881MALPVTALLLPLALLLHAARPqvqlqesgpglvkpsetlsltctvsgvslp dygvs wirqppgkgCAR 7 lewig viwgsettyyssslks rvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdy Full-aawgqgtlvtvssggggsggggsggggsggggseivmtqspatlslspgeratlsc rasqdiskylnwyqqkpgqaprlliy htsrlhs giparfsgsgsgtdytltisslqpedfavyfc qqgntlpyt fgggtkleiktttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrykfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmgalppr (SEQ ID NO: 69) CAR 8 104882atggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgctcgcccaca CAR 8-agtccagcttcaagaatcagggcctggtctggtgaagccatctgagactctgtccctcacttgcaFull-ntccgtgagcggagtgtccctcccagactacggagtgagctggattagacagcctcccggaaagggactggagtggatcggagtgatttggggtagcgaaaccacttactatcaatcttccctgaagtcacgggtcaccatttcaaaggataactcaaagaatcaagtgagcctcaagctctcatcagtcaccgccgctgacaccgccgtgtattactgtgccaagcattactactatggagggtcctacgccatggactactggggccagggaactctggtcactgtgtcatctggtggaggaggtagcggaggaggcgggagcggtggaggtggctccggaggcggtgggtcagaaatcgtgatgacccagagccctgcaaccctgtccctttctcccggggaacgggctaccctttcttgtcgggcatcacaagatatctcaaaatacctcaattggtatcaacagaagccgggacaggcccctaggcttcttatctaccacacctctcgcctgcatagcgggattcccgcacgctttagcgggtctggaagcgggaccgactacactctgaccatctcatctctccagcccgaggacttcgccgtctacttctgccagcagggtaacaccctgccgtacaccttcggccagggcaccaagcttgagatcaaaaccactactcccgctccaaggccacccacccctgccccgaccatcgcctctcagccgctttccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg (SEQ ID NO: 70) 104882MALPVTALLLPLALLLHAARPqvqlqesgpglvkpsetlsltctvsgvslp dygvs wirqppgkgCAR 8- lewig viwgsettyyqsslks rvtiskdnskngvslklssvtaadtavyycakhyyyggsyamdy Full-aawgqgtlvtvssggggsggggsggggsggggseivmtqspatlslspgeratlsc rasqdiskylnwyqqkpgqaprlliy htsrlhs giparfsgsgsgtdytltisslqpedfavyfc qqgntlpyt fgqgtkleiktttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (SEQ ID NO: 71) CAR 9 105974atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccga CAR 9aattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccctgtcttFull-ntgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagcggaggcggtgggagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactacaactcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg (SEQ ID NO: 72) 105974MALPVTALLLPLALLLHAARPeivmtqspatlslspgeratlsc rasqdiskyln wyqqkpgqapCAR 9- rlliy htsrlhs giparfsgsgsgtdytltisslqpedfavyfc qqgntlpytfgqgtkleikgg Full-aa ggsggggsggggsggggsqlqlqesgpglvkpsetlsltctvsgvslpdygvs wirqppgkglew ig viwgsettyynsslks rvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdy wgqgtlvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttgeedgcscrfpeeeeggcelrykfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr (SEQ ID NO: 73) CAR10 105975atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccga CAR 10aattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccctgtcttFull-ntgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagcggaggcggtgggagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactacaactcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg (SEQ ID NO: 74) 105975MALPVTALLLPLALLLHAARPEIVMTQSPATLSLSPGERATLSC RASQDISKYLN WYQQKPGQAPCAR 10 RLLIY HTSRLHS GIPARFSGSGSGTDYTLTISSLQPEDFAVYFC QQGNTLPYTFGQGTKLEIKGG Full-aa GGSGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVS WIRQPPGKGLEW IG VIWGSETTYYNSSLKS RVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDY WGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 75) CAR11 105976atggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgctcgcccaca CAR 11agtccagcttcaagaatcagggcctggtctggtgaagccatctgagactctgtccctcacttgcaFull-ntccgtgagcggagtgtccctcccagactacggagtgagctggattagacagcctcccggaaagggactggagtggatcggagtgatttggggtagcgaaaccacttactataactcttccctgaagtcacgggtcaccatttcaaaggataactcaaagaatcaagtgagcctcaagctctcatcagtcaccgccgctgacaccgccgtgtattactgtgccaagcattactactatggagggtcctacgccatggactactggggccagggaactctggtcactgtgtcatctggtggaggaggtagcggaggaggcgggagcggtggaggtggctccggaggtggcggaagcgaaatcgtgatgacccagagccctgcaaccctgtccctttctcccggggaacgggctaccctttcttgtcgggcatcacaagatatctcaaaatacctcaattggtatcaacagaagccgggacaggcccctaggcttcttatctaccacacctctcgcctgcatagcgggattcccgcacgctttagcgggtctggaagcgggaccgactacactctgaccatctcatctctccagcccgaggacttcgccgtctacttctgccagcagggtaacaccctgccgtacaccttcggccagggcaccaagcttgagatcaaaaccactactcccgctccaaggccacccacccctgccccgaccatcgcctctcagccgctttccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg (SEQ ID NO: 76) 105976MALPVTALLLPLALLLHAARPQVQLQESGPGLVKPSETLSLTCTVSGVSLP DYGVS WIRQPPGKGCAR 11 LEWIG VIWGSETTYYNSSLKS RVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDY Full-aaWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVMTQSPATLSLSPGERATLSC RASQDISKYLNWYQQKPGQAPRLLIY HTSRLHS GIPARFSGSGSGTDYTLTISSLQPEDFAVYFC QQGNTLPYT FGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 77) CAR12 105977atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggcccgaCAR 12-aattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccctgtcttFull-ntgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactacaactcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg (SEQ ID NO: 78) 105977MALPVTALLLPLALLLHAARPEIVMTQSPATLSLSPGERATLSC RASQDISKYLN WYQQKPGQAPCAR 12- RLLIY HTSRLHS GIPARFSGSGSGTDYTLTISSLQPEDFAVYFC QQGNTLPYTFGQGTKLEIKGG Full-aa GGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLP DYGVSWIRQPPGKGLEWIG VIW GSETTYYNSSLKS RVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDY WGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 79) CTL019 CTL019atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggaFull-ntcatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggaccaagctggagatcacaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggccaaggaacctcagtcaccgtctcctcaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc (SEQ ID NO: 80) CTL019MALPVTALLLPLALLLHAARPdiqmtqttsslsaslgdrvtiscrasqdiskylnwyqqkpdgtvFull-aaklliyhtsrlhsgvpsrfsgsgsgtdysltisnleqediatyfcqqgntlpytfgggtkleitggggsggggsggggsevklqesgpglvapsqslsvtctvsgvslpdygvswirqpprkglewlgviwgsettyynsalksrltiikdnsksqvflkmnslqtddtaiyycakhyyyggsyamdywgqgtsvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkgpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmgalppr (SEQ ID NO: 81)CD19 Associated Diseases and/or Disorders

In one aspect, the disclosure provides methods for treating cancer,e.g., a cancer associated with CD19 expression, with a CAR-expressingcell (e.g., T cell, NK cell) therapy. Exemplary cancers include, but arenot limited to e.g., one or more acute leukemias including but notlimited to, e.g., B-cell acute lymphocytic leukemia (“B-ALL”), T-cellacute lymphocytic leukemia (“T-ALL”), acute lymphocytic leukemia (ALL);one or more chronic leukemias including but not limited to, e.g.,chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL).Additional cancers or hematologic conditions associated with expressionof CD19 include, but are not limited to, e.g., B cell promyelocyticleukemia, blastic plasmacytoid dendritic cell neoplasm, Burkittslymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cellleukemia, small cell- or a large cell-follicular lymphoma, malignantlymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma(MCL), marginal zone lymphoma, multiple myeloma, myelodysplasia andmyelodysplastic syndrome, non-Hodgkin lymphoma, Hodgkin lymphoma,plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm,Waldenstrom macroglobulinemia, and “preleukemia” which are a diversecollection of hematological conditions united by ineffective production(or dysplasia) of myeloid blood cells, and the like. Further, a diseaseassociated with CD19 expression include, but not limited to, e.g.,atypical and/or non-classical cancers, malignancies, precancerousconditions or proliferative diseases associated with expression of CD19.

In one embodiment, the disclosure provides methods for treating CLL.

In another embodiment, the disclosure provides methods for treating ALL.

In another embodiment, the disclosure provides methods for treatingB-cell ALL.

In one aspect, the disclosure provides methods of treating a responder(e.g., a complete responder and partial responder) having cancer (e.g.,a hematological cancer such as ALL and CLL) with a CAR-expressing cell(e.g., T cell, NK cell) (e.g., a CD19 CAR-expressing cell (e.g., T cell,NK cell) as described herein, such as, e.g., CTL019). In an embodiment,the disclosure provides methods of treating a responder (e.g., acomplete responder and partial responder) with a CAR-expressing cell(e.g., T cell, NK cell) in combination with another therapeutic agent,e.g., another therapeutic agent described herein (e.g., another CAR,e.g., another CAR described herein, an inhibitory CAR, e.g., aninhibitory CAR described herein, a kinase inhibitor (e.g., a kinaseinhibitor described herein, e.g., an mTOR inhibitor, a BTK inhibitor), acheckpoint inhibitor, e.g., a checkpoint inhibitor described herein, astandard of care therapy, etc.). The combination can be, e.g., with anyagent described herein. In an embodiment, after a CAR-expressing cell(e.g., T cell, NK cell) treatment, e.g., an initial CAR-expressing cell(e.g., T cell, NK cell) treatment, a partial responder is tested by anyone of the methods described herein, such as, e.g., a CD19CAR-expressing cell (e.g., T cell, NK cell) gene set signature, and ifstatus has not changed and/or is down-graded to, e.g., a non-responder,then the subject is administered an alternative therapy, e.g., astandard of care for the particular cancer.

In one aspect, the disclosure provides methods of treating anon-responder having cancer (e.g., a hematological cancer such as ALLand CLL) with a CAR-expressing cell (e.g., T cell, NK cell) (e.g., aCD19 CAR-expressing cell (e.g., T cell, NK cell) as described herein,such as, e.g., CTL019). In an embodiment, the disclosure providesmethods of treating a non-responder with a CAR-expressing cell (e.g., Tcell, NK cell) in combination with another therapeutic agent, e.g.,another therapeutic agent described herein (e.g., another CAR, e.g.,another CAR described herein, an inhibitory CAR, e.g., an inhibitory CARdescribed herein, a kinase inhibitor (e.g., a kinase inhibitor describedherein, e.g., an mTOR inhibitor, a BTK inhibitor), a checkpointinhibitor, e.g., a checkpoint inhibitor described herein, a standard ofcare therapy, etc.). The combination can be, e.g., with any agentdescribed herein. In an embodiment, after a CAR-expressing cell (e.g., Tcell, NK cell) treatment, e.g., an initial CAR-expressing cell (e.g., Tcell, NK cell) treatment, a non-responder is tested by any one of themethods described herein, such as, e.g., a CD19 CAR-expressing cell(e.g., T cell, NK cell) gene set signature, and if status has changedand/or is up-graded to, e.g., a partial-responder, e.g., a completeresponder, then the subject is administered an alternative therapydescribed herein.

In an embodiment, the disclosure provides methods of treating cancer(e.g., a hematological cancer such as ALL or CLL) comprising steps of:(1) identifying a partial responder subject and/or non-respondersubject, (2) administering to the partial responder subject and/ornon-responder subject an mTOR inhibitor described herein, such as, e.g.,RAD001 and rapamycin, e.g., at a dose and/or dosing schedule describedherein; and (3) administering a CAR (e.g., a CD19 CAR described herein,such as, e.g., CTL019), e.g., subsequent to the administration of themTOR inhibitor, thus treating the cancer. In an embodiment, the methodfurther includes administering the mTOR inhibitor and/or the CAR incombination with one or more checkpoint inhibitors described here, suchas, e.g., a PD1inhibitor.

In an embodiment, the disclosure provides methods of treating cancer(e.g., a hematological cancer such as ALL or CLL) comprising steps of:(1) identifying a partial responder subject (e.g., patient) and/ornon-responder subject (e.g., patient), (2) enriching the T cellpopulation of the partial responder subject and/or non-responder subjectby selecting for a less exhausted and/or more naïve T cell population,(3) introducing (e.g., by transforming, transducing, infecting,electroporating, etc.) a CAR (e.g., a CD19 CAR described herein, suchas, e.g., CTL019) into said enriched T cell population thus transformingthe subject's T cell population; and (4) administering theCAR-expressing T cell population into the partial responder subjectand/or non-responder subject, thus treating the cancer.

In an embodiment, the disclosure provides methods of treating cancer(e.g., a hematological cancer such as ALL or CLL) comprising steps of:(1) identifying a partial responder subject (e.g., patient) and/ornon-responder subject (e.g., patient), (2) reevaluating a partialresponder subject and/or non-responder subject (e.g., patient) at alater time period for naïve T cells and/or less exhausted phenotype, and(3), e.g., if the subject has an increase in naïve T cells and/or a lessexhausted phenotype, administering a CAR-expressing cell (e.g., T cell,NK cell) therapy (e.g., a CD19 CAR-expressing cell (e.g., T cell, NKcell) as described herein, such as, e.g., CTL019), thus treating thecancer. In an embodiment, a later time period comprises at least 1 hour,at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours,at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours,at least 10 hours, at least 11 hours, at least 12 hours, at least 1 day,at least 2 days, at least 3 days, at least 4 days, at least 5 days, atleast 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, atleast 1 month, at least 2 months, at least 3 months, at least 4 months,at least 5 months, at least 6 months, at least 7 months at least 8months, at least 9 months, at least 10 months, at least 11 months, atleast 1 year, at least 2 years, at least 3 years, at least 4 years, atleast 5 years, at least 6 years, at least 7 years, at least 8 years, atleast 9 years, at least 10 years, or 11 years or more.

In an embodiment, the disclosure provides methods of treating cancer(e.g., a hematological cancer such as ALL or CLL) comprising the stepsof (1) identifying partial responders and/or non-responders; and (2)treating with an alternative therapy, e.g., a standard of care for theparticular cancer (e.g., the standard of care for ALL or CLL). In anembodiment, a partial responder is treated only with the standard ofcare (e.g., the standard of care for a hematological cancer such as ALLor CLL) in the absence of treatment with a CAR. In an embodiment, anon-responder is treated only with the standard of care (e.g., thestandard of care for a hematological cancer such as ALL or CLL) in theabsence of treatment with a CAR.

In an embodiment, standard of care for CLL includes, but is not limitedto exemplary therapies described herein, e.g., described in Table A, andcombinations thereof.

TABLE A Exemplary therapies for CLL w/o del (11q) or del del del(17p)(17p) (11q) First line ≥70 yrs with comorbidities Obinutuzumab +chlorambucil X X X Rituxan + chlorambucil X X Rituxan X Chlorambucil XFludarabine ± Rituxan X X Cladribine X Bendamustine ± Rituxan X X PCR(pentostatin, cyclophosphamide, Rituxan) X X First Line <70 yearswithout significant comorbidities FCR (Fludarabine, cyclophosphamide,Rituxan) X X X FR (Fludarabine, Rituxan) X X PCR X X Bendamustine ±Rituxan X X Obinutuzumab + chlorambucil X X X Second line-Relapsed/Refractory ≥70 years Imbruvica X X X Reduced-dose FCR X XReduced-dose PCRR X X Bendamustine ± Rituxan X X Ofatumumab X X XAlemutuzumab + Rituxan X X X High dose methylprednisone (HDMP) +rituximab X X X Lenalidomide + Rituxan X X X Dose dense rituximab X XSecond line- Relapsed/Refractory < years without significantcomorbiditites Imbruvica X X X FCR (Fludarabine, cyclophosphaide,Rituxan) X X PCR X X Bendamustine ± Rituxan X X Fludarabine +alemtuzumab X X R-CHOP (Rituxan, cyclophosphamide, X X X dosorubicin,vincristine, prednisone) Ofatumumab X X X OFAR (oxaliplatin, Fludara,cytarabine, Rituxan) X X X HDMP + rituximab X X X Lenalidomide + RituxanX X X

In an embodiment, standard of care for CLL includes (1) radiationtherapy, (2) chemotherapy, (3) surgery (e.g., removal of the spleen),(4) targeted therapy, (5) stem cell transplantation, and combinationsthereof. In an embodiment, the standard of care comprises externalradiation therapy. In an embodiment, the standard of care comprisesinternal radiation therapy (e.g., a radioactive substance sealed inneedles, wires or catheters, for example, that are placed directly intoor near the cancer).

In an embodiment, standard of care for ALL includes, but is not limitedto exemplary therapies described herein, e.g., described in Table B, andcombinations thereof.

TABLE B Exemplary therapies for ALL First Line RCHOP (Rituxan,cyclophosphamide, doxorubicin, vincristine, prednisone) Dose dense RCHOP14 (category 3) Dose adjusted EPOCH (etoposide, prednisone, vincristine,cyclophosphamide, doxorubicin) + Rituxan First Line Therapy for subjectswith Poor left ventricular function or very frail RCEPP (rituximab,cyclophosphamide, etoposide, prednisone, procarbazine) RCEOP (rituximab,cyclophosphamide, etoposide, vincristine, prednisone) RCNOP (rituximab,cyclophosphamide, mitoxantrone, vincristine, prednisone) RCEOP(rituximab, cyclophosphamide, etoposide, vincristine, prednisone) Doseadjusted EPOCH (etoposide, prednisone, vincristine, cyclophosphamide,doxorubicin) + Rituxan Second line- proceed to high dose therapy withautologous stem cell rescue DHAP (dexamethasone, cisplatin, cytarabine)± Rituxan ESHAP (etoposide, methylprednisolone, cytarabine, cisplatin) ±Rituxan GDP (gemcitabine, dexamethasone, cisplatin) ± RituxanGemOx(gemcitabine, oxaliplatin) ± Rituxan ICE (ifosfamide, carboplatin,etoposide) + Rituxan MINE (mesna, ifosfamide, mitoxantrone, etoposide) ±Rituxan Second-line therapy (non-candidates for high-dose therapy) CEPP(cyclophosphamide, etoposide, prednisone, procarbazine) ± Rituxan CEOP(cyclophosphamide, etoposide, vincristine, prednisone) ± RituxanDA-EPOCH ± Rituxan Revlimid ± Rituxan Rituxan GemOx ± Rituxan GDP ±Rituxan Bendamustine + Rituxan

In an embodiment, standard of care for ALL includes (1) chemotherapy,(2) radiation therapy, (3) stem cell transplantation, (4) biologicaltherapy, (5) targeted therapy, and combinations thereof.

In an embodiment, the standard of care includes, but is not limited to,fludarabine with cyclophosphamide (FC); fludarabine with rituximab (FR);fludarabine, cyclophosphamide, and rituximab (FCR); cyclophosphamide,doxorubicin, vincristine and prednisone (CHOP); and combinationsthereof. General chemotherapeutic agents considered for use include, butare not limited to anastrozole (Arimidex®), bicalutamide (Casodex®),bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection(Busulfex®), capecitabine (Xeloda®),N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®),carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®),cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®),cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposomeinjection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin(Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®),daunorubicin citrate liposome injection (DaunoXome®), dexamethasone,docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®),etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil(Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, Gemcitabine(difluorodeoxycitidine), hydroxyurea (Hydrea®), Idarubicin (Idamycin®),ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®),leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine(Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®),mylotarg, paclitaxel (Taxol®), phoenix (Yttrium90/MX-DTPA), pentostatin,polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate(Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine(Tirazone®), topotecan hydrochloride for injection (Hycamptin®),vinblastine (Velban®), vincristine (Oncovin®), vinorelbine (Navelbine®),and combinations thereof.

In an embodiment, chemotherapy comprises an antimetabolite, including,but not limited to, folic acid antagonists (also referred to herein asantifolates), pyrimidine analogs, purine analogs and adenosine deaminaseinhibitors): methotrexate (Rheumatrex®, Trexall®), 5-fluorouracil(Adrucil®, Efudex®, Fluoroplex®), floxuridine (FUDF®), cytarabine(Cytosar-U®, Tarabine PFS), 6-mercaptopurine (Puri-Nethol®)),6-thioguanine (Thioguanine Tabloid®), fludarabine phosphate (Fludara®),pentostatin (Nipent®), pemetrexed (Alimta®), raltitrexed (Tomudex®),cladribine (Leustatin®), clofarabine (Clofarex®, Clolar®), cytarabineliposomal (also known as Liposomal Ara-C, DepoCyt™); decitabine(Dacogen®); hydroxyurea (Hydrea®, Droxia™ and Mylocel™); mercaptopurine(Puri-Nethol®), pralatrexate (Folotyn™) capecitabine (Xeloda®),nelarabine (Arranon®), azacitidine (Vidaza®) and gemcitabine (Gemzar®).Suitable antimetabolites include, e.g., 5-fluorouracil (Adrucil®,Efudex®, Fluoroplex®), floxuridine (FUDF®), capecitabine (Xeloda®),pemetrexed (Alimta®), raltitrexed (Tomudex®) and gemcitabine (Gemzar®),and combinations thereof. In an embodiment, the purine analogue isfludarabine.

In an embodiment, chemotherapy comprises an alkylating agent including,but not limited to nitrogen mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas and triazenes, uracil mustard (AminouracilMustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®,Haemanthamine®, Nordopan®, Uracil nitrogen Mustard®, Uracillost®,Uracilmostaza®, Uramustin®, Uramustine®), chlormethine (Mustargen®),cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®,Revimmune™), ifosfamide (Mitoxana®), melphalan (Alkeran®), Chlorambucil(Leukeran®), pipobroman (Amedel®, Vercyte®), triethylenemelamine(Hemel®, Hexalen®, Hexastat®), triethylenethiophosphoramine,Temozolomide (Temodar®), thiotepa (Thioplex®), busulfan (Busilvex®,Myleran®), carmustine (BiCNU®), lomustine (CeeNU®), streptozocin(Zanosar®), and Dacarbazine (DTIC-Dome®) and combinations thereof.Additional exemplary alkylating agents include, without limitation,Oxaliplatin (Eloxatin®); Temozolomide (Temodar® and Temodal®);Dactinomycin (also known as actinomycin-D, Cosmegen®); Melphalan (alsoknown as L-PAM, L-sarcolysin, and phenylalanine mustard, Alkeran®);Altretamine (also known as hexamethylmelamine (HMM), Hexalen®);Carmustine (BiCNU®); Bendamustine (Treanda®); Busulfan (Busulfex® andMyleran®); Carboplatin (Paraplatin®); Lomustine (also known as CCNU,CeeNU®); Cisplatin (also known as CDDP, Platinol® and Platinol®-AQ);Chlorambucil (Leukeran®); Cyclophosphamide (Cytoxan® and Neosar®);Dacarbazine (also known as DTIC, DIC and imidazole carboxamide,DTIC-Dome®); Altretamine (also known as hexamethylmelamine (HMM),Hexalen®); Ifosfamide (Ifex®); Prednumustine; Procarbazine (Matulane®);Mechlorethamine (also known as nitrogen mustard, mustine andmechloroethamine hydrochloride, Mustargen®); Streptozocin (Zanosar®);Thiotepa (also known as thiophosphoamide, TESPA and TSPA, Thioplex®);Cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®);Bendamustine HCl (Treanda®) and combinations thereof. In an embodiment,the alkylating agent is bendamustine. In an embodiment, the alkylatingagent is cyclophosphamide.

In an embodiment, the chemotherapeutic agent is a kinase inhibitor,e.g., a tyrosine kinase inhibitor including, but not limited to,erlotinib hydrochloride (Tarceva®); linifanib(N-[4-(3-amino-1H-indazol-4-yl)phenyl]-N-2-fluoro-5-methylphenyl)urea,also known as ABT 869, available from Genentech); sunitinib malate(Sutent®); bosutinib(4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4-methylpiperazin-1-yl)propoxy]quinoline-3-carbonitrile,also known as SKI-606, and described in U.S. Pat. No. 6,780,996);dasatinib (Sprycel®); pazopanib (Votrient®); sorafenib (Nexavar®);zactima (ZD6474); and imatinib or imatinib mesylate (Gilvec® andGleevec®). In one embodiment, the kinase inhibitor is a BTK inhibitorselected from ibrutinib (PCI-32765); GDC-0834; RN-486; CGI-560;CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; and LFM-A13. In oneembodiment, the kinase inhibitor is a CDK4 inhibitor selected fromaloisine A; flavopiridol or HMR-1275,2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4-chromenone;crizotinib (PF-02341066;2-(2-Chlorophenyl)-5,7-dihydroxy-8-[(2R,3S)-2-(hydroxymethyl)-1-methyl-3-pyrrolidinyl]-4H-1-benzopyran-4-one,hydrochloride (P276-00);1-methyl-5-[[2-[5-(trifluoromethyl)-1H-imidazol-2-yl]-4-pyridinyl]oxy]-N-[4-(trifluoromethyl)phenyl]-1H-benzimidazol-2-amine(RAF265); indisulam (E7070); roscovitine (CYC202); palbociclib(PD0332991); dinaciclib (SCH727965);N-[5-[[(5-tert-butyloxazol-2-yl)methyl]thio]thiazol-2-yl]piperidine-4-carboxamide(BMS 387032); 4-[[9-chloro-7-(2,6-difluorophenyl)-5H-1.5pyrimido[5,4-d][2]benzazepin-2-yl]amino]-benzoic acid (MLN8054);5-[3-(4,6-difluoro-1H-benzimidazol-2-yl)-1H-indazol-5-yl]-N-ethyl-4-methyl-3-pyridinemethanamine(AG-024322); 4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acidN-(piperidin-4-yl)amide (AT7519);4-[2-methyl-1-(1-methylethyl)-1H-imidazol-5-yl]-N-[4-(methylsulfonyl)phenyl]-2-pyrimidinamine(AZD5438); and XL281 (BMS908662). In one embodiment, the kinaseinhibitor is an MNK inhibitor selected from CGP052088;4-amino-3-(p-fluorophenylamino)-pyrazolo [3,4-d] pyrimidine (CGP57380);cercosporamide; ETC-1780445-2; and 4-amino-5-(4-fluoroanilino)-pyrazolo[3,4-d] pyrimidine.

In an embodiment, targeted therapy includes, but is not limited to ananti-CD20 antibody or functional fragment thereof, such as, e.g.,rituximab (Riuxan® and MabThera®); tositumomab (Bexxar®); and ofatumumab(Arzerra®), and combinations thereof. In one embodiment, the targetedtherapy includes, but is not limited to, an anti-CD52 antibody orfunctional fragment thereof such as, e.g., alemtuzumab (Campath®).

In an embodiment, biologic therapy comprises immunotherapy. Exemplaryanthracyclines include, without limitation, doxorubicin (Adriamycin® andRubex®); bleomycin (Lenoxane®); daunorubicin (dauorubicin hydrochloride,daunomycin, and rubidomycin hydrochloride, Cerubidine®); daunorubicinliposomal (daunorubicin citrate liposome, DaunoXome®); mitoxantrone(DHAD, Novantrone®); epirubicin (Ellence™); idarubicin (Idamycin®,Idamycin PFS®); mitomycin C (Mutamycin®); geldanamycin; herbimycin;ravidomycin; desacetylravidomycin and combinations thereof.

In an embodiment, stem cell transplantation comprises an autogeneic stemcell transplant. In an embodiment, stem cell transplantation comprisesan allogenic stem cell transplant. In an embodiment, stem celltransplantation comprises allogeneic bone marrow transplantation. In anembodiment, stem cell transplantation comprises a hematopoietic stemcell transplantation (HSCT). In an embodiment, hematopoietic stem cellsare derived from various tissues including, but not limited to bonemarrow, peripheral blood, umbilical cord blood, and combinationsthereof.

In an embodiment, the provided methods comprise determining if thesubject is identified as having a statistically significant differencein expression level of one or more markers listed in Table 1A, Table 1B,Table 7A, Table 7B, Table 8, Table 9, Table 10, Table 14, Table 15 andTable 16, or a PD-1 biomarker, LAG-3 biomarker, TIM-3 biomarker, CD57biomarker CD27 biomarker, CD122 biomarker, CD62L biomarker and a KLRG1biomarker, relative to a reference level, and administering to thesubject a therapeutically effective dose of a CAR-expressing cell, e.g.,a T cell or NK cell. In an embodiment, a CAR-expressing cell (e.g., Tcell, NK cell) is a CD19 CAR-expressing cell (e.g., T cell, NK cell)described herein such as, e.g., CTL019.

In one aspect, the disclosure provides methods for treating a diseaseassociated with CD19 expression. In one aspect, the invention providesmethods for treating a disease wherein part of the tumor is negative forCD19 and part of the tumor is positive for CD19. For example, providedmethods are useful for treating subjects that have undergone treatmentfor a disease associated with elevated expression of CD19, wherein thesubject that has undergone treatment for elevated levels of CD19exhibits a disease associated with elevated levels of CD19.

In one aspect, provided methods comprise a vector comprising CD19 CARoperably linked to promoter for expression in mammalian cells (e.g., Tcells or NK cells). In one aspect, provided methods comprise arecombinant cell (e.g., T cell or NK cell) expressing a CD19 CAR for usein treating CD19-expressing tumors, wherein the recombinant T cellexpressing the CD19 CAR is termed a CD19 CAR-expressing cell. In oneaspect, a CD19 CAR-expressing cell (e.g., T cell, NK cell) administeredaccording to provided methods is capable of contacting a tumor cell withat least one CD19 CAR expressed on its surface such that theCAR-expressing cell targets the tumor cell and growth of the tumor isinhibited.

In one aspect, the disclosure features to a method of inhibiting growthof a CD19-expressing tumor cell, comprising contacting the tumor cellwith a CD19 CAR-expressing cell (e.g., T cell, NK cell) described hereinsuch that the CAR-expressing cell is activated in response to theantigen and targets the cancer cell, wherein the growth of the tumor isinhibited.

In one aspect, the disclosure includes a type of cellular therapy whereT cells are genetically modified to express a CAR and the CAR-expressingcell (e.g., T cell, NK cell) is infused to a recipient in need thereof.The infused cell is able to kill tumor cells in the recipient. Unlikeantibody therapies, CAR-modified cells (e.g., T cells or NK cells) areable to replicate in vivo resulting in long-term persistence that canlead to sustained tumor control. In various aspects, the cellsadministered to the patient, or their progeny, persist in the patientfor at least four months, five months, six months, seven months, eightmonths, nine months, ten months, eleven months, twelve months, thirteenmonths, fourteen month, fifteen months, sixteen months, seventeenmonths, eighteen months, nineteen months, twenty months, twenty-onemonths, twenty-two months, twenty-three months, two years, three years,four years, or five years after administration of the cell to thepatient.

The disclosure also includes a type of cellular therapy where cells(e.g., T cells, NK cells) are modified, e.g., by in vitro transcribedRNA, to transiently express a chimeric antigen receptor (CAR) and theCAR-expressing cell (e.g., T cell, NK cell) is infused to a recipient inneed thereof. The infused cell is able to kill tumor cells in therecipient. Thus, in various aspects, the cells administered to thepatient, are present for less than one month, e.g., three weeks, twoweeks, one week, after administration of the cell (e.g., T cell, NKcell) to the patient.

Without wishing to be bound by any particular theory, the anti-tumorimmunity response elicited by the CAR-modified cells (e.g., T cells, NKcells) may be an active or a passive immune response, or alternativelymay be due to a direct vs indirect immune response. In one aspect, theCAR transduced T cells exhibit specific proinflammatory cytokinesecretion and potent cytolytic activity in response to human cancercells expressing the CD19, resist soluble CD19 inhibition, mediatebystander killing and mediate regression of an established human tumor.For example, antigen-less tumor cells within a heterogeneous field ofCD19-expressing tumor may be susceptible to indirect destruction byCD19-redirected T cells that has previously reacted against adjacentantigen-positive cancer cells.

In one aspect, the fully-human CAR-modified cells (e.g., T cells, NKcells) described herein may be a type of vaccine for ex vivoimmunization and/or in vivo therapy in a mammal. In one aspect, themammal is a human.

With respect to ex vivo immunization, at least one of the followingoccurs in vitro prior to administering the cell into a subject: i)expansion of the cells, ii) introducing a nucleic acid encoding a CAR tothe cells or iii) cryopreservation of the cells.

Ex vivo procedures are well known in the art and are discussed morefully below. Briefly, cells are isolated from a subject (e.g., a human)and genetically modified (i.e., transduced or transfected in vitro) witha vector expressing a CAR disclosed herein. The CAR-modified cell can beadministered to a mammalian recipient to provide a therapeutic benefit.The mammalian recipient may be a human and the CAR-modified cell can beautologous with respect to the recipient. Alternatively, the cells canbe allogeneic, syngeneic or xenogeneic with respect to the recipient.

Hematologic Cancer

Hematological cancer conditions are types of cancer such as leukemia andmalignant lymphoproliferative conditions that affect blood, bone marrowand the lymphatic system.

Leukemia can be classified as acute leukemia and chronic leukemia. Acuteleukemia can be further classified as acute myelogenous leukemia (AML)and acute lymphoid leukemia (ALL). Chronic leukemia includes chronicmyelogenous leukemia (CML) and chronic lymphoid leukemia (CLL). Otherrelated conditions include myelodysplastic syndromes (MDS, formerlyknown as “preleukemia”) which are a diverse collection of hematologicalconditions united by ineffective production (or dysplasia) of myeloidblood cells and risk of transformation to AML.

Lymphoma is a group of blood cell tumors that develop from lymphocytes.Exemplary lymphomas include non-Hodgkin lymphoma and Hodgkin lymphoma.

The present disclosure provides for compositions and methods fortreating cancer. In one aspect, the cancer is a hematologic cancerincluding but is not limited to a leukemia or a lymphoma. In one aspect,the CAR-expressing cells (e.g., T cells, NK cells) of the invention maybe used to treat cancers and malignancies such as, but not limited to,e.g., acute leukemias including but not limited to, e.g., B-cell acutelymphoid leukemia (“B-ALL”), T-cell acute lymphoid leukemia (“T-ALL”),acute lymphoid leukemia (ALL); one or more chronic leukemias includingbut not limited to, e.g., chronic myelogenous leukemia (CML), chroniclymphocytic leukemia (CLL); additional hematologic cancers orhematologic conditions including, but not limited to, e.g., B cellpromyelocytic leukemia, blastic plasmacytoid dendritic cell neoplasm,Burkitts lymphoma, diffuse large B cell lymphoma, follicular lymphoma,hairy cell leukemia, small cell- or a large cell-follicular lymphoma,malignant lymphoproliferative conditions, MALT lymphoma, mantle celllymphoma (MCL), marginal zone lymphoma, multiple myeloma, myelodysplasiaand myelodysplastic syndrome, non-Hodgkin lymphoma, Hodgkin lymphoma,plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm,Waldenstrom macroglobulinemia, and “preleukemia” which are a diversecollection of hematological conditions united by ineffective production(or dysplasia) of myeloid blood cells, and the like. Further a diseaseassociated with CD19 expression includes, but not limited to, e.g.,atypical and/or non-classical cancers, malignancies, precancerousconditions or proliferative diseases expressing CD19.

The present disclosure also provides methods for inhibiting theproliferation or reducing a CD19-expressing cell population, the methodscomprising contacting a population of cells comprising a CD19-expressingcell with a CD19 CAR-expressing cell (e.g., T cell, NK cell) describedherein that binds to the CD19-expressing cell. In a specific aspect, thedisclosure provides methods for inhibiting the proliferation or reducingthe population of cancer cells expressing CD19, the methods comprisingcontacting the CD19-expressing cancer cell population with a CD19CAR-expressing cell (e.g., T cell, NK cell) described herein that bindsto the CD19-expressing cell. In one aspect, the present disclosureprovides methods for inhibiting the proliferation or reducing thepopulation of cancer cells expressing CD19, the methods comprisingcontacting the CD19-expressing cancer cell population with a CD19CAR-expressing cell (e.g., T cell, NK cell) described herein that bindsto the CD19-expressing cell. In certain aspects, the anti-CD19CAR-expressing cell (e.g., T cell, NK cell) reduces the quantity,number, amount or percentage of cells and/or cancer cells by at least25%, at least 30%, at least 40%, at least 50%, at least 65%, at least75%, at least 85%, at least 95%, or at least 99% in a subject with oranimal model for myeloid leukemia or another cancer associated withCD19-expressing cells relative to a negative control. In one aspect, thesubject is a human.

The present disclosure also provides methods for preventing, treatingand/or managing a disease associated with CD19-expressing cells (e.g., ahematologic cancer or atypical cancer expressing CD19), the methodscomprising administering to a subject in need a CAR-expressing cell(e.g., T cell, NK cell) described herein that binds to theCD19-expressing cell. In one aspect, the subject is a human.Non-limiting examples of disorders associated with CD19-expressing cellsinclude autoimmune disorders (such as lupus), inflammatory disorders(such as allergies and asthma) and cancers (such as hematologicalcancers or atypical cancers expressing CD19).

The present disclosure also provides methods for preventing, treatingand/or managing a disease associated with CD19-expressing cells, themethods comprising administering to a subject in need a CD19CAR-expressing cell (e.g., T cell, NK cell) described herein that bindsto the CD19-expressing cell. In one aspect, the subject is a human.

The present disclosure provides methods for preventing relapse of cancerassociated with CD19-expressing cells (e.g., a hematological cancer suchas ALL and CLL), the methods comprising administering to a subject inneed thereof a CD19 CAR-expressing cell (e.g., T cell, NK cell)described herein that binds to the CD19-expressing cell. In one aspect,the methods comprise administering to the subject in need thereof aneffective amount of a CD19 CAR-expressing cell (e.g., T cell, NK cell)described herein that binds to the CD19-expressing cell in combinationwith an effective amount of another therapy.

Combination Therapy

It will be appreciated that any cancer therapy as described above andherein, can be administered in combination with one or more additionaltherapies to treat and/or reduce the symptoms of cancer describedherein. The pharmaceutical compositions can be administered concurrentlywith, prior to, or subsequent to, one or more other additional therapiesor therapeutic agents. In an embodiment, a CAR-expressing cell describedherein may be used in combination with other known agents and therapies.Administered “in combination”, as used herein, means that two (or more)different treatments are delivered to the subject during the course ofthe subject affliction with the disorder, e.g., the two or moretreatments are delivered after the subjects has been diagnosed with thedisorder and before the disorder has been cured or eliminated ortreatment has ceased for other reasons. In some embodiments, thedelivery of one treatment is still occurring when the delivery of thesecond begins, so that there is overlap in terms of administration. Thisis sometimes referred to herein as “simultaneous” or “concurrentdelivery”. In other embodiments, the delivery of one treatment endsbefore the delivery of the other treatment begins. In some embodimentsof either case, the treatment is more effective because of combinedadministration. For example, the second treatment is more effective,e.g., an equivalent effect is seen with less of the second treatment, orthe second treatment reduces symptoms to a greater extent, than would beseen if the second treatment were administered in the absence of thefirst treatment or the analogous situation is seen with the firsttreatment. In some embodiments, delivery is such that the reduction in asymptom, or other parameter related to the disorder is greater than whatwould be observed with one treatment delivered in the absence of theother. The effect of the two treatments can be partially additive,wholly additive, or greater than additive. The delivery can be such thatan effect of the first treatment delivered is still detectable when thesecond is delivered.

A CAR-expressing cell described herein and the at least one additionaltherapeutic agent can be administered simultaneously, in the same or inseparate compositions, or sequentially. For sequential administration,the CAR-expressing cell described herein can be administered first, andthe additional agent can be administered second, or the order ofadministration can be reversed.

The CAR therapy and/or other therapeutic agents, procedures ormodalities can be administered during periods of active disorder, orduring a period of remission or less active disease. The CAR therapy canbe administered before the other treatment, concurrently with thetreatment, post-treatment, or during remission of the disorder.

When administered in combination, the CAR therapy and the additionalagent (e.g., second or third agent), or all, can be administered in anamount or dose that is higher, lower or the same than the amount ordosage of each agent used individually, e.g., as a monotherapy. Incertain embodiments, the administered amount or dosage of the CARtherapy, the additional agent (e.g., second or third agent), or all, islower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%)than the amount or dosage of each agent used individually, e.g., as amonotherapy. In other embodiments, the amount or dosage of the CARtherapy, the additional agent (e.g., second or third agent), or all,that results in a desired effect (e.g., treatment of cancer) is lower(e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower)than the amount or dosage of each agent used individually, e.g., as amonotherapy, required to achieve the same therapeutic effect.

In further aspects, a CAR-expressing cell described herein may be usedin a treatment regimen in combination with surgery, chemotherapy,radiation, immunosuppressive agents, such as cyclosporin, azathioprine,methotrexate, mycophenolate, and FK506, antibodies, or otherimmunoablative agents such as CAMPATH, anti-CD3 antibodies or otherantibody therapies, cytoxin, fludarabine, cyclosporin, FK506, rapamycin,mycophenolic acid, steroids, FR901228, cytokines, and irradiationpeptide vaccine, such as that described in Izumoto et al. 2008 JNEUROSURG 108:963-971.

In one embodiment, a CAR-expressing cell described herein can be used incombination with a chemotherapeutic agent. Exemplary chemotherapeuticagents include those described in paragraphs 873-874 of InternationalApplication WO2015/142675, filed Mar. 13, 2015, which is hereinincorporated by reference in its entirety, and combinations thereof.

Exemplary alkylating agents include, without limitation, those describedin paragraph 875 of International Application WO2015/142675, filed Mar.13, 2015, which is herein incorporated by reference in its entirety, andcombinations thereof.

Exemplary mTOR inhibitors include, without limitation, RAD001,temsirolimus; ridaforolimus (formally known as deferolimus,(1R,2R,4S)-4-[(2R)-2[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0^(4,9)]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669, and described inPCT Publication No. WO 03/064383); everolimus (Afinitor® or RAD001);rapamycin (AY22989, Sirolimus®); simapimod (CAS 164301-51-3);emsirolimus,(5-{2,4-Bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol(AZD8055);2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one(PF04691502, CAS 1013101-36-4); andN²-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-arginylglycyl-L-α-aspartylL-serine-(SEQID NO: 91), inner salt (SF1126, CAS 936487-67-1), XL765 and combinationsthereof.

Exemplary immunomodulators include, without limitation, those describedin paragraph 882 of International Application WO2015/142675, filed Mar.13, 2015, which is herein incorporated by reference in its entirety, andcombinations thereof.

Exemplary anthracyclines include, without limitation, those described inparagraph 883 of International Application WO2015/142675, filed Mar. 13,2015, which is herein incorporated by reference in its entirety, andcombinations thereof.

Exemplary vinca alkaloids include, without limitation, those describedin paragraph 884 of International Application WO2015/142675, filed Mar.13, 2015, which is herein incorporated by reference in its entirety, andcombinations thereof.

Exemplary proteosome inhibitors include, without limitation, thosedescribed in paragraph 884 of International Application WO2015/142675,filed Mar. 13, 2015, which is herein incorporated by reference in itsentirety, and combinations thereof.

In some embodiments, a CAR-expressing cell described herein isadministered in combination with an oncolytic virus. In embodiments,oncolytic viruses are capable of selectively replicating in andtriggering the death of or slowing the growth of a cancer cell. In somecases, oncolytic viruses have no effect or a minimal effect onnon-cancer cells. An oncolytic virus includes but is not limited to anoncolytic adenovirus, oncolytic Herpes Simplex Viruses, oncolyticretrovirus, oncolytic parvovirus, oncolytic vaccinia virus, oncolyticSinbis virus, oncolytic influenza virus, or oncolytic RNA virus (e.g.,oncolytic reovirus, oncolytic Newcastle Disease Virus (NDV), oncolyticmeasles virus, or oncolytic vesicular stomatitis virus (VSV)).

In an embodiment, cells expressing a CAR described herein areadministered to a subject in combination with a molecule that decreasesthe Treg cell population. Methods that decrease the number of (e.g.,deplete) Treg cells are known in the art and include, e.g., CD25depletion, cyclophosphamide administration, modulating GITR function.Without wishing to be bound by theory, it is believed that reducing thenumber of Treg cells in a subject prior to apheresis or prior toadministration of a CAR-expressing cell described herein reduces thenumber of unwanted immune cells (e.g., Tregs) in the tumormicroenvironment and reduces the subject's risk of relapse.

In one embodiment, a CAR expressing cell described herein areadministered to a subject in combination with a molecule targeting GITRand/or modulating GITR functions, such as a GITR agonist and/or a GITRantibody that depletes regulatory T cells (Tregs). In embodiments, cellsexpressing a CAR described herein are administered to a subject incombination with cyclophosphamide. In one embodiment, the GITR bindingmolecules and/or molecules modulating GITR functions (e.g., GITR agonistand/or Treg depleting GITR antibodies) are administered prior toadministration of the CAR-expressing cell. For example, in oneembodiment, the GITR agonist can be administered prior to apheresis ofthe cells. In embodiments, cyclophosphamide is administered to thesubject prior to administration (e.g., infusion or re-infusion) of theCAR-expressing cell or prior to apheresis of the cells. In embodiments,cyclophosphamide and an anti-GITR antibody are administered to thesubject prior to administration (e.g., infusion or re-infusion) of theCAR-expressing cell or prior to apheresis of the cells. In oneembodiment, the subject has cancer (e.g., a solid cancer or ahematological cancer such as ALL or CLL). In an embodiment, the subjecthas CLL. In embodiments, the subject has ALL. In embodiments, thesubject has a solid cancer, e.g., a solid cancer described herein.Exemplary GITR agonists include, without limitation, GITR fusionproteins and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies)such as, e.g., a GITR fusion protein described in U.S. Pat. No.6,111,090, European Patent No.: 090505B1, U.S. Pat. No. 8,586,023, PCTPublication Nos.: WO 2010/003118 and 2011/090754, or an anti-GITRantibody described, e.g., in U.S. Pat. No. 7,025,962, European PatentNo.: 1947183B1, U.S. Pat. Nos. 7,812,135, 8,388,967, 8,591,886, EuropeanPatent No.: EP 1866339, PCT Publication No.: WO 2011/028683, PCTPublication No.:WO 2013/039954, PCT Publication No.: WO2005/007190, PCTPublication No.: WO 2007/133822, PCT Publication No.: WO2005/055808, PCTPublication No.: WO 99/40196, PCT Publication No.: WO 2001/03720, PCTPublication No.: WO99/20758, PCT Publication No.: WO2006/083289, PCTPublication No.: WO 2005/115451, U.S. Pat. No. 7,618,632, and PCTPublication No.: WO 2011/051726.

In an embodiment, a CAR expressing cell described herein, such as, e.g.,a CD19 CAR-expressing cell (e.g., T cell, NK cell), e.g., CTL019, isadministered to a subject, e.g., a subject identified as a partialresponder or non-responder, in combination with a GITR agonist, e.g., aGITR agonist described herein. In an embodiment, the GITR agonist isadministered prior to the CAR-expressing cell. For example, in anembodiment, the GITR agonist can be administered prior to apheresis ofthe cells. In an embodiment, the subject has cancer (e.g., ahematological cancer such as ALL and CLL). In an embodiment, the subjecthas ALL. In an embodiment, the subject has CLL.

In an embodiment, a CAR expressing cell described herein, such as, e.g.,a CD19 CAR-expressing cell (e.g., T cell, NK cell), e.g., CTL019 isadministered to a subject, e.g., a subject identified as a partialresponder or non-responder, in combination with an mTOR inhibitor, e.g.,an mTOR inhibitor described herein, e.g., a target of the rapamycinsignaling pathway such as RAD001. In an embodiment, the mTOR inhibitoris administered prior to the CAR-expressing cell. For example, in anembodiment, the mTOR inhibitor can be administered prior to apheresis ofthe cells. In an embodiment, the subject has cancer (e.g., ahematological cancer such as ALL and CLL). In an embodiment, the subjecthas ALL. In an embodiment, the subject has CLL.

Kinase Inhibitor

In one embodiment, a CAR-expressing cell described herein may be used ina treatment regimen in combination with a kinase inhibitor, e.g., a CDK4inhibitor, a BTK inhibitor, an MNK inhibitor, an mTOR inhibitor, an ITKinhibitor, etc. In one embodiment, the subject is a complete responder,and the subject is administered a treatment regimen that includesadministration of a CAR-expressing cell described herein in combinationwith a kinase inhibitor, e.g., a kinase inhibitor described herein,e.g., at a dose or dosing schedule described herein. In one embodiment,the subject is a partial responder or a non-responder, and the subjectis administered a treatment regimen that includes administration of aCAR-expressing cell described herein in combination with a kinaseinhibitor, e.g., a kinase inhibitor described herein, e.g., at a dose ordosing schedule described herein.

In an embodiment, the kinase inhibitor is a CDK4 inhibitor, e.g., a CDK4inhibitor described herein, e.g., a CDK4/6 inhibitor, such as, e.g.,6-Acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one,hydrochloride (also referred to as palbociclib or PD0332991). In oneembodiment, the kinase inhibitor is a BTK inhibitor, e.g., a BTKinhibitor described herein, such as, e.g., ibrutinib. In one embodiment,the kinase inhibitor is an mTOR inhibitor, e.g., an mTOR inhibitordescribed herein, such as, e.g., rapamycin, a rapamycin analog, OSI-027.The mTOR inhibitor can be, e.g., an mTORC1 inhibitor and/or an mTORC2inhibitor, e.g., an mTORC1 inhibitor and/or mTORC2 inhibitor describedherein. In one embodiment, the kinase inhibitor is a MNK inhibitor,e.g., a MNK inhibitor described herein, such as, e.g.,4-amino-5-(4-fluoroanilino)-pyrazolo [3,4-d] pyrimidine. The MNKinhibitor can be, e.g., a MNKla, MNK1b, MNK2a and/or MNK2b inhibitor.

In one embodiment, the kinase inhibitor is a CDK4 inhibitor selectedfrom aloisine A; flavopiridol or HMR-1275,2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4-chromenone;crizotinib (PF-02341066;2-(2-Chlorophenyl)-5,7-dihydroxy-8-[(2R,3S)-2-(hydroxymethyl)-1-methyl-3-pyrrolidinyl]-4H-1-benzopyran-4-one,hydrochloride (P276-00);1-methyl-5-[[2-[5-(trifluoromethyl)-1H-imidazol-2-yl]-4-pyridinyl]oxy]-N-[4-(trifluoromethyl)phenyl]-1H-benzimidazol-2-amine(RAF265); indisulam (E7070); roscovitine (CYC202); palbociclib(PD0332991); dinaciclib (SCH727965);N-[5-[[(5-tert-butyloxazol-2-yl)methyl]thio]thiazol-2-yl]piperidine-4-carboxamide(BMS 387032);4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]-benzoicacid (MLN8054);5-[3-(4,6-difluoro-1H-benzimidazol-2-yl)-1H-indazol-5-yl]-N-ethyl-4-methyl-3-pyridinemethanamine(AG-024322); 4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acidN-(piperidin-4-yl)amide (AT7519);4-[2-methyl-1-(1-methylethyl)-1H-imidazol-5-yl]-N-[4-(methylsulfonyl)phenyl]-2-pyrimidinamine(AZD5438); and XL281 (BMS908662).

In one embodiment, the kinase inhibitor is a CDK4 inhibitor, e.g.,palbociclib (PD0332991), and the palbociclib is administered at a doseof about 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 105 mg, 110mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg (e.g., 75 mg, 100 mg or 125mg) daily for a period of time, e.g., daily for 14-21 days of a 28 daycycle, or daily for 7-12 days of a 21 day cycle. In one embodiment, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of palbociclib areadministered.

In one embodiment, the kinase inhibitor is a BTK inhibitor selected fromibrutinib (PCI-32765); GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224;CC-292; ONO-4059; CNX-774; and LFM-A13.

In one embodiment, the kinase inhibitor is a BTK inhibitor, e.g.,ibrutinib (PCI-32765), and the ibrutinib is administered at a dose ofabout 250 mg, 300 mg, 350 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg,500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600 mg (e.g., 250 mg, 420 mg or560 mg) daily for a period of time, e.g., daily for 21 day cycle, ordaily for 28 day cycle. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12 or more cycles of ibrutinib are administered.

In one embodiment, the kinase inhibitor is an mTOR inhibitor selectedfrom temsirolimus; ridaforolimus (1R,2R,4S)-4-[(2R)-2[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0^(4,9)]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669; everolimus(RAD001); rapamycin (AY22989); simapimod;(5-{2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol(AZD8055);2-mmino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one(PF04691502); andN²-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]L-arginylglycyl-L-α-aspartylL-serine-,inner salt (SF1126); and XL765.

In one embodiment, the kinase inhibitor is an mTOR inhibitor, e.g.,rapamycin, and the rapamycin is administered at a dose of about 3 mg, 4mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg (e.g., 6 mg) daily for a periodof time, e.g., daily for 21 day cycle, or daily for 28 day cycle. In oneembodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles ofrapamycin are administered. In one embodiment, the kinase inhibitor isan mTOR inhibitor, e.g., everolimus and the everolimus is administeredat a dose of about 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg (e.g., 10 mg) daily for aperiod of time, e.g., daily for 28 day cycle. In one embodiment, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of everolimus areadministered.

In one embodiment, the kinase inhibitor is an MNK inhibitor selectedfrom CGP052088; 4-amino-3-(p-fluorophenylamino)-pyrazolo [3,4-d]pyrimidine (CGP57380); cercosporamide; ETC-1780445-2; and4-amino-5-(4-fluoroanilino)-pyrazolo [3,4-d] pyrimidine.

In some embodiments of the methods, uses, and compositions herein, theBTK inhibitor is a BTK inhibitor described in International ApplicationWO/2015/079417, which is herein incorporated by reference in itsentirety. For instance, in some embodiments, the BTK inhibitor is acompound of formula (I) or a pharmaceutically acceptable salt thereof;

wherein,

R1 is hydrogen, C1-C6 alkyl optionally substituted by hydroxy;

R2 is hydrogen or halogen;

R3 is hydrogen or halogen;

R4 is hydrogen;

R5 is hydrogen or halogen;

or R4 and R5 are attached to each other and stand for a bond, —CH2-,—CH2-CH2-, —CH═CH—, —CH═CH—CH2-; —CH2-CH═CH—; or —CH2-CH2-CH2-;

R6 and R7 stand independently from each other for H, C1-C6 alkyloptionally substituted by hydroxyl, C3-C6 cycloalkyl optionallysubstituted by halogen or hydroxy, or halogen;

R8, R9, R, R′, R10 and R11 independently from each other stand for H, orC1-C6 alkyl optionally substituted by C1-C6 alkoxy; or any two of R8,R9, R, R′, R10 and R11 together with the carbon atom to which they arebound may form a 3-6 membered saturated carbocyclic ring;

R12 is hydrogen or C1-C6 alkyl optionally substituted by halogen orC1-C6 alkoxy;

or R12 and any one of R8, R9, R, R′, R10 or R11 together with the atomsto which they are bound may form a 4, 5, 6 or 7 membered azacyclic ring,which ring may optionally be substituted by halogen, cyano, hydroxyl,C1-C6 alkyl or C1-C6 alkoxy;

n is 0 or 1; and

R13 is C2-C6 alkenyl optionally substituted by C1-C6 alkyl, C1-C6 alkoxyor N,N-di-C1-C6 alkyl amino; C2-C6 alkynyl optionally substituted byC1-C6 alkyl or C1-C6 alkoxy; or C2-C6 alkylenyl oxide optionallysubstituted by C1-C6 alkyl.

Low, Immune Enhancing, Dose of an mTOR Inhibitor

Methods described herein can use a low, immune enhancing, dose of anmTOR inhibitor e.g., an allosteric mTOR inhibitor, including rapalogssuch as RAD001. Administration of a low, immune enhancing, dose of anmTOR inhibitor (e.g., a dose that is insufficient to completely suppressthe immune system, but sufficient to improve immune function) canoptimize the performance of immune effector cells, e.g., T cells orCAR-expressing cells, in the subject. Methods for measuring mTORinhibition, dosages, treatment regimens, and suitable pharmaceuticalcompositions are described in U.S. Patent Application No. 2015/0140036,filed Nov. 13, 2014, hereby incorporated by reference.

In an embodiment, administration of a low, immune enhancing, dose of anmTOR inhibitor can result in one or more of the following:

-   -   i) a decrease in the number of PD-1 positive immune effector        cells;    -   ii) an increase in the number of PD-1 negative immune effector        cells;    -   iii) an increase in the ratio of PD-1 negative immune effector        cells/PD-1 positive immune effector cells;    -   iv) an increase in the number of naive T cells;    -   v) an increase in the expression of one or more of the following        markers: CD62L^(high), CD127^(high), CD27⁺, and BCL2, e.g., on        memory T cells, e.g., memory T cell precursors;    -   vi) a decrease in the expression of KLRG1, e.g., on memory T        cells, e.g., memory T cell precursors; or    -   vii) an increase in the number of memory T cell precursors,        e.g., cells with any one or combination of the following        characteristics: increased CD62L^(high), increased CD127^(high),        increased CD27⁺, decreased KLRG1, and increased BCL2;        and wherein any of the foregoing, e.g., i), ii), iii), iv), v),        vi), or vii), occurs e.g., at least transiently, e.g., as        compared to a non-treated subject.

In another embodiment, administration of a low, immune enhancing, doseof an mTOR inhibitor results in increased or prolonged proliferation ofCAR-expressing cells, e.g., in culture or in a subject, e.g., ascompared to non-treated CAR-expressing cells or a non-treated subject.In embodiments, increased proliferation is associated with in anincrease in the number of CAR-expressing cells. In another embodiment,administration of a low, immune enhancing, dose of an mTOR inhibitorresults in increased killing of cancer cells by CAR-expressing cells,e.g., in culture or in a subject, e.g., as compared to non-treatedCAR-expressing cells or a non-treated subject. In embodiments, increasedkilling of cancer cells is associated with in a decrease in tumorvolume.

In one embodiment, the cells expressing a CAR molecule, e.g., a CARmolecule described herein, are administered in combination with a low,immune enhancing dose of an mTOR inhibitor, e.g., an allosteric mTORinhibitor, e.g., RAD001, or a catalytic mTOR inhibitor. For example,administration of the low, immune enhancing, dose of the mTOR inhibitorcan be initiated prior to administration of a CAR-expressing celldescribed herein; completed prior to administration of a CAR-expressingcell described herein; initiated at the same time as administration of aCAR-expressing cell described herein; overlapping with administration ofa CAR-expressing cell described herein; or continuing afteradministration of a CAR-expressing cell described herein.

Alternatively or in addition, administration of a low, immune enhancing,dose of an mTOR inhibitor can optimize immune effector cells to beengineered to express a CAR molecule described herein. In suchembodiments, administration of a low, immune enhancing, dose of an mTORinhibitor, e.g., an allosteric inhibitor, e.g., RAD001, or a catalyticinhibitor, is initiated or completed prior to harvest of immune effectorcells, e.g., T cells or NK cells, to be engineered to express a CARmolecule described herein, from a subject.

In another embodiment, immune effector cells, e.g., T cells or NK cells,to be engineered to express a CAR molecule described herein, e.g., afterharvest from a subject, or CAR-expressing immune effector cells, e.g., Tcells or NK cells, e.g., prior to administration to a subject, can becultured in the presence of a low, immune enhancing, dose of an mTORinhibitor.

In an embodiment, administering to the subject a low, immune enhancing,dose of an mTOR inhibitor comprises administering, e.g., once per week,e.g., in an immediate release dosage form, 0.1 to 20, 0.5 to 10, 2.5 to7.5, 3 to 6, or about 5, mgs of RAD001, or a bioequivalent dose thereof.In an embodiment, administering to the subject a low, immune enhancing,dose of an mTOR inhibitor comprises administering, e.g., once per week,e.g., in a sustained release dosage form, 0.3 to 60, 1.5 to 30, 7.5 to22.5, 9 to 18, or about 15 mgs of RAD001, or a bioequivalent dosethereof.

In an embodiment, a dose of an mTOR inhibitor is associated with, orprovides, mTOR inhibition of at least 5 but no more than 90%, at least10 but no more than 90%, at least 15, but no more than 90%, at least 20but no more than 90%, at least 30 but no more than 90%, at least 40 butno more than 90%, at least 50 but no more than 90%, at least 60 but nomore than 90%, at least 70 but no more than 90%, at least 5 but no morethan 80%, at least 10 but no more than 80%, at least 15, but no morethan 80%, at least 20 but no more than 80%, at least 30 but no more than80%, at least 40 but no more than 80%, at least 50 but no more than 80%,at least 60 but no more than 80%, at least 5 but no more than 70%, atleast 10 but no more than 70%, at least 15, but no more than 70%, atleast 20 but no more than 70%, at least 30 but no more than 70%, atleast 40 but no more than 70%, at least 50 but no more than 70%, atleast 5 but no more than 60%, at least 10 but no more than 60%, at least15, but no more than 60%, at least 20 but no more than 60%, at least 30but no more than 60%, at least 40 but no more than 60%, at least 5 butno more than 50%, at least 10 but no more than 50%, at least 15, but nomore than 50%, at least 20 but no more than 50%, at least 30 but no morethan 50%, at least 40 but no more than 50%, at least 5 but no more than40%, at least 10 but no more than 40%, at least 15, but no more than40%, at least 20 but no more than 40%, at least 30 but no more than 40%,at least 35 but no more than 40%, at least 5 but no more than 30%, atleast 10 but no more than 30%, at least 15, but no more than 30%, atleast 20 but no more than 30%, or at least 25 but no more than 30%.

The extent of mTOR inhibition can be conveyed as, or corresponds to, theextent of P70 S6 kinase inhibition, e.g., the extent of mTOR inhibitioncan be determined by the level of decrease in P70 S6 kinase activity,e.g., by the decrease in phosphorylation of a P70 S6 kinase substrate.The level of mTOR inhibition can be evaluated by various methods, suchas measuring P70 S6 kinase activity by the Boulay assay, as described inU.S. Patent Application No. 2015/01240036, hereby incorporated byreference, or as described in U.S. Pat. No. 7,727,950, herebyincorporated by reference; measuring the level of phosphorylated S6 bywestern blot; or evaluating a change in the ratio of PD1 negative immuneeffector cells to PD1 positive immune effector cells.

As used herein, the term “mTOR inhibitor” refers to a compound orligand, or a pharmaceutically acceptable salt thereof, which inhibitsthe mTOR kinase in a cell. In an embodiment, an mTOR inhibitor is anallosteric inhibitor. Allosteric mTOR inhibitors include the neutraltricyclic compound rapamycin (sirolimus), rapamycin-related compounds,that is compounds having structural and functional similarity torapamycin including, e.g., rapamycin derivatives, rapamycin analogs(also referred to as rapalogs) and other macrolide compounds thatinhibit mTOR activity. In an embodiment, an mTOR inhibitor is acatalytic inhibitor.

Rapamycin is a known macrolide antibiotic produced by Streptomyceshygroscopicus having the structure shown in Formula A.

See, e.g., McAlpine, J. B., et al., J. Antibiotics (1991) 44: 688;Schreiber, S. L., et al., J. Am. Chem. Soc. (1991) 113: 7433; U.S. Pat.No. 3,929,992. There are various numbering schemes proposed forrapamycin. To avoid confusion, when specific rapamycin analogs are namedherein, the names are given with reference to rapamycin using thenumbering scheme of formula A.

Rapamycin analogs useful in the invention are, for example,O-substituted analogs in which the hydroxyl group on the cyclohexyl ringof rapamycin is replaced by OR₁ in which R₁ is hydroxyalkyl,hydroxyalkoxyalkyl, acylaminoalkyl, or aminoalkyl; e.g. RAD001, alsoknown as, everolimus as described in U.S. Pat. No. 5,665,772 andWO94/09010 the contents of which are incorporated by reference. Othersuitable rapamycin analogs include those substituted at the 26- or28-position. The rapamycin analog may be an epimer of an analogmentioned above, particularly an epimer of an analog substituted inposition 40, 28 or 26, and may optionally be further hydrogenated, e.g.as described in U.S. Pat. No. 6,015,815, WO95/14023 and WO99/15530 thecontents of which are incorporated by reference, e.g. ABT578 also knownas zotarolimus or a rapamycin analog described in U.S. Pat. No.7,091,213, WO98/02441 and WO01/14387 the contents of which areincorporated by reference, e.g. AP23573 also known as ridaforolimus.

Examples of rapamycin analogs suitable for use in the present inventionfrom U.S. Pat. No. 5,665,772 include, but are not limited to,40-O-benzyl-rapamycin, 40-O-(4′-hydroxymethyl)benzyl-rapamycin,40-O-[4′-(1,2-dihydroxyethyl)]benzyl-rapamycin, 40-O-allyl-rapamycin,40-O-[3′-(2,2-dimethyl-1,3-dioxolan-4(S)-yl)-prop-2′-en-1′-yl]-rapamycin,(2′E,4'S)-40-O-(4′,5′-dihydroxypent-2′-en-1′-yl)-rapamycin,40-O-(2-hydroxy)ethoxycarbonylmethyl-rapamycin,40-O(2-hydroxy)ethyl-rapamycin, 40-O-(3-hydroxy)propyl-rapamycin,40-O(6-hydroxy)hexyl-rapamycin,40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin,40-O-[(3S)-2,2-dimethyldioxolan-3-yl]methyl-rapamycin,40-O-[(2S)-2,3-dihydroxyprop-1-yl]-rapamycin,40-O-(2-acetoxy)ethyl-rapamycin, 40-O-(2-nicotinoyloxy)ethyl-rapamycin,40-O[2-(N-morpholino)acetoxy]ethyl-rapamycin,40-O-(2-N-imidazolylacetoxy)ethyl-rapamycin,40-O-[2-(N-methyl-N′-piperazinyl)acetoxy]ethyl-rapamycin,39-O-desmethyl-39,40-O,O-ethylene-rapamycin,(26R)-26-dihydro-40-O-(2-hydroxy)ethyl-rapamycin,40-O(2-aminoethyl)-rapamycin, 40-O(2-acetaminoethyl)-rapamycin,40-O(2-nicotinamidoethyl)-rapamycin,40-O-(2-N-methyl-imidazo-2′-ylcarbethoxamido)ethyl)-rapamycin,40-O-(2-ethoxycarbonylaminoethyl)-rapamycin,40-O-(2-tolylsulfonamidoethyl)-rapamycin and40-O-[2-4′,5′-dicarboethoxy-1′,2′,3′-triazol-1′-yl)-ethyl]-rapamycin.

Other rapamycin analogs useful in the present invention are analogswhere the hydroxyl group on the cyclohexyl ring of rapamycin and/or thehydroxy group at the 28 position is replaced with an hydroxyester groupare known, for example, rapamycin analogs found in U.S. RE44,768, e.g.temsirolimus.

Other rapamycin analogs useful in the preset invention include thosewherein the methoxy group at the 16 position is replaced with anothersubstituent, preferably (optionally hydroxy-substituted) alkynyloxy,benzyl, orthomethoxybenzyl or chlorobenzyl and/or wherein the mexthoxygroup at the 39 position is deleted together with the 39 carbon so thatthe cyclohexyl ring of rapamycin becomes a cyclopentyl ring lacking the39 position methyoxy group; e.g. as described in WO95/16691 andWO96/41807 the contents of which are incorporated by reference. Theanalogs can be further modified such that the hydroxy at the 40-positionof rapamycin is alkylated and/or the 32-carbonyl is reduced.

Rapamycin analogs from WO95/16691 include, but are not limited to,16-demthoxy-16-(pent-2-ynyl)oxy-rapamycin,16-demthoxy-16-(but-2-ynyl)oxy-rapamycin,16-demthoxy-16-(propargyl)oxy-rapamycin,16-demethoxy-16-(4-hydroxy-but-2-ynyl)oxy-rapamycin,16-demthoxy-16-benzyloxy-40-O-(2-hydroxyethyl)-rapamycin,16-demthoxy-16-benzyloxy-rapamycin,16-demethoxy-16-ortho-methoxybenzyl-rapamycin,16-demethoxy-40-O-(2-methoxyethyl)-16-pent-2-ynyl)oxy-rapamycin,39-demethoxy-40-desoxy-39-formyl-42-nor-rapamycin,39-demethoxy-40-desoxy-39-hydroxymethyl-42-nor-rapamycin,39-demethoxy-40-desoxy-39-carboxy-42-nor-rapamycin,39-demethoxy-40-desoxy-39-(4-methyl-piperazin-1-yl)carbonyl-42-nor-rapamycin,39-demethoxy-40-desoxy-39-(morpholin-4-yl)carbonyl-42-nor-rapamycin,39-demethoxy-40-desoxy-39-[N-methyl,N-(2-pyridin-2-yl-ethyl)]carbamoyl-42-nor-rapamycin and39-demethoxy-40-desoxy-39-(p-toluenesulfonylhydrazonomethyl)-42-nor-rapamycin.

Rapamycin analogs from WO96/41807 include, but are not limited to,32-deoxo-rapamycin, 16-O-pent-2-ynyl-32-deoxo-rapamycin,16-O-pent-2-ynyl-32-deoxo-40-O-(2-hydroxy-ethyl)-rapamycin,16-O-pent-2-ynyl-32-(S)-dihydro-40-O-(2-hydroxyethyl)-rapamycin,32(S)-dihydro-40-O-(2-methoxy)ethyl-rapamycin and32(S)-dihydro-40-O-(2-hydroxyethyl)-rapamycin.

Another suitable rapamycin analog is umirolimus as described inUS2005/0101624 the contents of which are incorporated by reference.

RAD001, otherwise known as everolimus (Afinitor®), has the chemical name(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18-dihydroxy-12-{(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl}-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-aza-tricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20-pentaone

Further examples of allosteric mTOR inhibitors include sirolimus(rapamycin, AY-22989),40-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]-rapamycin (alsocalled temsirolimus or CCI-779) and ridaforolimus (AP-23573/MK-8669).Other examples of allosteric mTor inhibitors include zotarolimus(ABT578) and umirolimus.

Alternatively or additionally, catalytic, ATP-competitive mTORinhibitors have been found to target the mTOR kinase domain directly andtarget both mTORC1 and mTORC2. These are also more effective inhibitorsof mTORC1 than such allosteric mTOR inhibitors as rapamycin, becausethey modulate rapamycin-resistant mTORC1 outputs such as 4EBP1-T37/46phosphorylation and cap-dependent translation.

Catalytic inhibitors include: BEZ235 or2-methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)-phenyl]-propionitrile,or the monotosylate salt form. the synthesis of BEZ235 is described inWO2006/122806; CCG168 (otherwise known as AZD-8055, Chresta, C. M., etal., Cancer Res, 2010, 70(1), 288-298) which has the chemical name{5-[2,4-bis-((S)-3-methyl-morpholin-4-yl)-pyrido[2,3d]pyrimidin-7-yl]-2-methoxy-phenyl}-methanol; 3-[2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl]-N-methylbenzamide(WO09104019);3-(2-aminobenzo[d]oxazol-5-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine(WO10051043 and WO2013023184); AN-(3-(N-(3-((3,5-dimethoxyphenyl)amino)quinoxaline-2-yl)sulfamoyl)phenyl)-3-methoxy-4-methylbenzamide(WO07044729 and WO12006552); PKI-587 (Venkatesan, A. M., J. Med. Chem.,2010, 53, 2636-2645) which has the chemical name1-[4-[4-(dimethylamino)piperidine-1-carbonyl]phenyl]-3-[4-(4,6-dimorpholino-1,3,5-triazin-2-yl)phenyl]urea;GSK-2126458 (ACS Med. Chem. Lett., 2010, 1, 39-43) which has thechemical name2,4-difluoro-N-{2-methoxy-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}benzenesulfonamide;5-(9-isopropyl-8-methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine(WO10114484);(E)-N-(8-(6-amino-5-(trifluoromethyl)pyridin-3-yl)-1-(6-(2-cyanopropan-2-yl)pyridin-3-yl)-3-methyl-1H-imidazo[4,5-c]quinolin-2(3H)-ylidene)cyanamide(WO12007926).

Further examples of catalytic mTOR inhibitors include8-(6-methoxy-pyridin-3-yl)-3-methyl-1-(4-piperazin-1-yl-3-trifluoromethyl-phenyl)-1,3-dihydro-imidazo[4,5-c]quinolin-2-one(WO2006/122806) and Ku-0063794 (Garcia-Martinez J M, et al., Biochem J.,2009, 421(1), 29-42. Ku-0063794 is a specific inhibitor of the mammaliantarget of rapamycin (mTOR).) WYE-354 is another example of a catalyticmTor inhibitor (Yu K, et al. (2009). Biochemical, Cellular, and In vivoActivity of Novel ATP-Competitive and Selective Inhibitors of theMammalian Target of Rapamycin. Cancer Res. 69(15): 6232-6240).

mTOR inhibitors useful according to the present invention also includeprodrugs, derivatives, pharmaceutically acceptable salts, or analogsthereof of any of the foregoing.

mTOR inhibitors, such as RAD001, may be formulated for delivery based onwell-established methods in the art based on the particular dosagesdescribed herein. In particular, U.S. Pat. No. 6,004,973 (incorporatedherein by reference) provides examples of formulations useable with themTOR inhibitors described herein.

Inhibitory Molecule Inhibitors/Checkpoint Inhibitors

In one embodiment, the subject can be administered an agent whichenhances the activity of a CAR-expressing cell. For example, in oneembodiment, the agent can be an agent which inhibits a checkpointmolecule. Checkpoint molecules, e.g., Programmed Death 1 (PD1), can, insome embodiments, decrease the ability of a CAR-expressing cell to mountan immune effector response. Examples of inhibitory molecules, e.g.,checkpoint molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM(e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT,LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM(TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9,adenosine, and TGFR (e.g., TGFRbeta). In embodiments, the CAR-expressingcell described herein comprises a switch costimulatory receptor, e.g.,as described in WO 2013/019615, which is incorporated herein byreference in its entirety.

The methods described herein can include administration of aCAR-expressing cell in combination with a checkpoint inhibitor. In oneembodiment, the subject is a complete responder. In another embodiment,the subject is a partial responder or non-responder, and, e.g., in someembodiments, the checkpoint inhibitor is administered prior to theCAR-expressing cell, e.g., two weeks, 12 days, 10 days, 8 days, oneweek, 6 days, 5 days, 4 days, 3 days, 2 days or 1 day beforeadministration of the CAR-expressing cell. In some embodiments, thecheckpoint inhibitor is administered concurrently with theCAR-expressing cell.

Inhibition of a checkpoint molecule, e.g., by inhibition at the DNA, RNAor protein level, can optimize a CAR-expressing cell performance. Inembodiments, an inhibitory nucleic acid, e.g., an inhibitory nucleicacid, e.g., a dsRNA, e.g., a siRNA or shRNA, a clustered regularlyinterspaced short palindromic repeats (CRISPR), atranscription-activator like effector nuclease (TALEN), or a zinc fingerendonuclease (ZFN), e.g., as described herein, can be used to inhibitexpression of a checkpoint molecule in the CAR-expressing cell. In anembodiment, the inhibitor is a shRNA. In an embodiment, the checkpointmolecule is inhibited within a CAR-expressing cell. In theseembodiments, a dsRNA molecule that inhibits expression of the checkpointmolecule is linked to the nucleic acid that encodes a component, e.g.,all of the components, of the CAR.

In one embodiment, the inhibitor of an inhibitory signal can be, e.g.,an antibody or antibody fragment that binds to a checkpoint molecule.For example, the agent can be an antibody or antibody fragment thatbinds to PD1, PD-L1, PD-L2 or CTLA4 (e.g., ipilimumab (also referred toas MDX-010 and MDX-101, and marketed as Yervoy®; Bristol-Myers Squibb;Tremelimumab (IgG2 monoclonal antibody available from Pfizer, formerlyknown as ticilimumab, CP-675,206). In an embodiment, the agent is anantibody or antibody fragment that binds to TIM3. In an embodiment, theagent is an antibody or antibody fragment that binds to LAG3. In anembodiment, the agent is an antibody or antibody fragment that binds toCEACAM.

PD1 is an inhibitory member of the CD28 family of receptors that alsoincludes CD28, CTLA-4, ICOS, and BTLA. PD1 is expressed on activated Bcells, T cells and myeloid cells (Agata et al. 1996 INT. IMMUNOL8:765-75). Two ligands for PD1, PD-L1 and PD-L2 have been shown todownregulate T cell activation upon binding to PD1 (Freeman et a. 2000 JExp Med 192:1027-34; Latchman et al. 2001 NAT IMMUNOL 2:261-8; Carter etal. 2002 EUR J IMMUNOL 32:634-43). PD-L1 is abundant in human cancers(Dong et al. 2003 J MOL MED 81:281-7; Blank et al. 2005 CANCER IMMUNOL.IMMUNOTHER. 54:307-314; Konishi et al. 2004 CLIN CANCER RES 10:5094).Immune suppression can be reversed by inhibiting the local interactionof PD1 with PD-L1.

Antibodies, antibody fragments, and other inhibitors of PD1, PD-L1 andPD-L2 are available in the art and may be used combination with a CARdescribed herein, e.g., a CD19 CAR described herein. For example,nivolumab (also referred to as BMS-936558 or MDX1106; Bristol-MyersSquibb) is a fully human IgG4 monoclonal antibody which specificallyblocks PD1. Nivolumab (clone 5C4) and other human monoclonal antibodiesthat specifically bind to PD1 are disclosed in U.S. Pat. No. 8,008,449and WO2006/121168. Pidilizumab (CT-011; Cure Tech) is a humanized IgGlkmonoclonal antibody that binds to PD1Pidilizumab and other humanizedanti-PD1 monoclonal antibodies are disclosed in WO2009/101611.Lambrolizumab (also referred to as MK03475; Merck) is a humanized IgG4monoclonal antibody that binds to PD1. Lambrolizumab and other humanizedanti-PD1 antibodies are disclosed in U.S. Pat. No. 8,354,509 andWO2009/114335. MDPL3280A (Genentech/Roche) is a human Fc optimized IgG1monoclonal antibody that binds to PD-L1. MDPL3280A and other humanmonoclonal antibodies to PD-L1 are disclosed in U.S. Pat. No. 7,943,743and U.S Publication No.: 20120039906. Other anti-PD-L1 binding agentsinclude YW243.55.S70 (heavy and light chain variable regions are shownin SEQ ID NOs 20 and 21 in WO2010/077634) and MDX-1 105 (also referredto as BMS-936559, and, e.g., anti-PD-L1 binding agents disclosed inWO2007/005874). AMP-224 (B7-DCIg; Amplimmune; e.g., disclosed inWO2010/027827 and WO2011/066342), is a PD-L2 Fc fusion soluble receptorthat blocks the interaction between PD1 and B7-H1. Other anti-PD1antibodies include AMP 514 (Amplimmune), among others, e.g., anti-PD1antibodies disclosed in U.S. Pat. No. 8,609,089, US 2010028330, and/orUS 20120114649.

In one embodiment, the anti-PD-1 antibody or fragment thereof is ananti-PD-1 antibody molecule as described in US 2015/0210769, entitled“Antibody Molecules to PD-1 and Uses Thereof,” incorporated by referencein its entirety. In one embodiment, the anti-PD-1 antibody moleculeincludes at least one, two, three, four, five or six CDRs (orcollectively all of the CDRs) from a heavy and light chain variableregion from an antibody chosen from any of BAP049-hum01, BAP049-hum02,BAP049-hum03, BAP049-hum04, BAP049-hum05, BAP049-hum06, BAP049-hum07,BAP049-hum08, BAP049-hum09, BAP049-hum10, BAP049-hum11, BAP049-hum12,BAP049-hum13, BAP049-hum14, BAP049-hum15, BAP049-hum16, BAP049-Clone-A,BAP049-Clone-B, BAP049-Clone-C, BAP049-Clone-D, or BAP049-Clone-E; or asdescribed in Table 1 of US 2015/0210769, or encoded by the nucleotidesequence in Table 1, or a sequence substantially identical (e.g., atleast 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to anyof the aforesaid sequences; or closely related CDRs, e.g., CDRs whichare identical or which have at least one amino acid alteration, but notmore than two, three or four alterations (e.g., substitutions,deletions, or insertions, e.g., conservative substitutions).

In yet another embodiment, the anti-PD-1 antibody molecule comprises atleast one, two, three or four variable regions from an antibodydescribed herein, e.g., an antibody chosen from any of BAP049-hum01,BAP049-hum02, BAP049-hum03, BAP049-hum04, BAP049-hum05, BAP049-hum06,BAP049-hum07, BAP049-hum08, BAP049-hum09, BAP049-hum10, BAP049-hum11,BAP049-hum12, BAP049-hum13, BAP049-hum14, BAP049-hum15, BAP049-hum16,BAP049-Clone-A, BAP049-Clone-B, BAP049-Clone-C, BAP049-Clone-D, orBAP049-Clone-E; or as described in Table 1 of US 2015/0210769, orencoded by the nucleotide sequence in Table 1; or a sequencesubstantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%,98%, 99% or higher identical) to any of the aforesaid sequences.

TIM3 (T cell immunoglobulin-3) also negatively regulates T cellfunction, particularly in IFN-g-secreting CD4+T helper 1 and CD8+Tcytotoxic 1 cells, and plays a critical role in T cell exhaustion.Inhibition of the interaction between TIM3 and its ligands, e.g.,galectin-9 (Gal9), phosphotidylserine (PS), and HMGB1, can increaseimmune response. Antibodies, antibody fragments, and other inhibitors ofTIM3 and its ligands are available in the art and may be usedcombination with a CD19 CAR described herein. For example, antibodies,antibody fragments, small molecules, or peptide inhibitors that targetTIM3 binds to the IgV domain of TIM3 to inhibit interaction with itsligands. Antibodies and peptides that inhibit TIM3 are disclosed inWO2013/006490 and US20100247521. Other anti-TIM3 antibodies includehumanized versions of RMT3-23 (disclosed in Ngiow et al., 2011, CancerRes, 71:3540-3551), and clone 8B.2C12 (disclosed in Monney et al., 2002,Nature, 415:536-541). Bi-specific antibodies that inhibit TIM3 and PD-1are disclosed in US20130156774.

In one embodiment, the anti-TIM3 antibody or fragment thereof is ananti-TIM3 antibody molecule as described in US 2015/0218274, entitled“Antibody Molecules to TIM3 and Uses Thereof,” incorporated by referencein its entirety. In one embodiment, the anti-TIM3 antibody moleculeincludes at least one, two, three, four, five or six CDRs (orcollectively all of the CDRs) from a heavy and light chain variableregion from an antibody chosen from any of ABTIM3, ABTIM3-hum01,ABTIM3-hum02, ABTIM3-hum03, ABTIM3-hum04, ABTIM3-hum05, ABTIM3-hum06,ABTIM3-hum07, ABTIM3-hum08, ABTIM3-hum09, ABTIM3-hum10, ABTIM3-hum11,ABTIM3-hum12, ABTIM3-hum13, ABTIM3-hum14, ABTIM3-hum15, ABTIM3-hum16,ABTIM3-hum17, ABTIM3-hum18, ABTIM3-hum19, ABTIM3-hum20, ABTIM3-hum21,ABTIM3-hum22, ABTIM3-hum23; or as described in Tables 1-4 of US2015/0218274; or encoded by the nucleotide sequence in Tables 1-4; or asequence substantially identical (e.g., at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or higher identical) to any of the aforesaidsequences, or closely related CDRs, e.g., CDRs which are identical orwhich have at least one amino acid alteration, but not more than two,three or four alterations (e.g., substitutions, deletions, orinsertions, e.g., conservative substitutions).

In yet another embodiment, the anti-TIM3 antibody molecule comprises atleast one, two, three or four variable regions from an antibodydescribed herein, e.g., an antibody chosen from any of ABTIM3,ABTIM3-hum01, ABTIM3-hum02, ABTIM3-hum03, ABTIM3-hum04, ABTIM3-hum05,ABTIM3-hum06, ABTIM3-hum07, ABTIM3-hum08, ABTIM3-hum09, ABTIM3-hum10,ABTIM3-hum11, ABTIM3-hum12, ABTIM3-hum13, ABTIM3-hum14, ABTIM3-hum15,ABTIM3-hum16, ABTIM3-hum17, ABTIM3-hum18, ABTIM3-hum19, ABTIM3-hum20,ABTIM3-hum21, ABTIM3-hum22, ABTIM3-hum23; or as described in Tables 1-4of US 2015/0218274; or encoded by the nucleotide sequence in Tables 1-4;or a sequence substantially identical (e.g., at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaidsequences.

In other embodiments, the agent which enhances the activity of aCAR-expressing cell is a CEACAM inhibitor (e.g., CEACAM-1, CEACAM-3,and/or CEACAM-5 inhibitor). In one embodiment, the inhibitor of CEACAMis an anti-CEACAM antibody molecule. Exemplary anti-CEACAM-1 antibodiesare described in WO 2010/125571, WO 2013/082366 WO 2014/059251 and WO2014/022332, e.g., a monoclonal antibody 34B1, 26H7, and 5F4; or arecombinant form thereof, as described in, e.g., US 2004/0047858, U.S.Pat. No. 7,132,255 and WO 99/052552. In other embodiments, theanti-CEACAM antibody binds to CEACAM-5 as described in, e.g., Zheng etal. PLoS One. 2010 Sep. 2; 5(9). pii: e12529(DOI:10:1371/journal.pone.0021146), or crossreacts with CEACAM-1 andCEACAM-5 as described in, e.g., WO 2013/054331 and US 2014/0271618.

Without wishing to be bound by theory, carcinoembryonic antigen celladhesion molecules (CEACAM), such as CEACAM-1 and CEACAM-5, are believedto mediate, at least in part, inhibition of an anti-tumor immuneresponse (see e.g., Markel et al. J Immunol. 2002 Mar. 15;168(6):2803-10; Markel et al. J Immunol. 2006 Nov. 1; 177(9):6062-71;Markel et al. Immunology. 2009 February; 126(2):186-200; Markel et al.Cancer Immunol Immunother. 2010 February; 59(2):215-30; Ortenberg et al.Mol Cancer Ther. 2012 June; 11(6):1300-10; Stern et al. J Immunol. 2005Jun. 1; 174(11):6692-701; Zheng et al. PLoS One. 2010 Sep. 2; 5(9). pii:e12529). For example, CEACAM-1 has been described as a heterophilicligand for TIM-3 and as playing a role in TIM-3-mediated T celltolerance and exhaustion (see e.g., WO 2014/022332; Huang, et al. (2014)Nature doi:10.1038/nature13848). In embodiments, co-blockade of CEACAM-1and TIM-3 has been shown to enhance an anti-tumor immune response inxenograft colorectal cancer models (see e.g., WO 2014/022332; Huang, etal. (2014), supra). In other embodiments, co-blockade of CEACAM-1 andPD-1 reduce T cell tolerance as described, e.g., in WO 2014/059251.Thus, CEACAM inhibitors can be used with the other immunomodulatorsdescribed herein (e.g., anti-PD-1 and/or anti-TIM-3 inhibitors) toenhance an immune response against a cancer, e.g., a melanoma, a lungcancer (e.g., NSCLC), a bladder cancer, a colon cancer an ovariancancer, and other cancers as described herein.

LAG3 (lymphocyte activation gene-3 or CD223) is a cell surface moleculeexpressed on activated T cells and B cells that has been shown to play arole in CD8+ T cell exhaustion. Antibodies, antibody fragments, andother inhibitors of LAG3 and its ligands are available in the art andmay be used combination with a CD19 CAR described herein. For example,BMS-986016 (Bristol-Myers Squib) is a monoclonal antibody that targetsLAG3. IMP701 (Immutep) is an antagonist LAG3 antibody and IMP731(Immutep and GlaxoSmithKline) is a depleting LAG3 antibody. Other LAG3inhibitors include IMP321 (Immutep), which is a recombinant fusionprotein of a soluble portion of LAG3 and Ig that binds to MHC class IImolecules and activates antigen presenting cells (APC). Other antibodiesare disclosed, in WO2010/019570.

In some embodiments, the agent which enhances the activity of aCAR-expressing cell can be, e.g., a fusion protein comprising a firstdomain and a second domain, wherein the first domain is a checkpointmolecule, or fragment thereof, and the second domain is a polypeptidethat is associated with a positive signal, e.g., a polypeptidecomprising an intracellular signaling domain as described herein (alsoreferred to herein as an inhibitory CAR or iCAR). In some embodiments,the polypeptide that is associated with a positive signal can include acostimulatory domain of CD28, CD27, ICOS, e.g., an intracellularsignaling domain of CD28, CD27 and/or ICOS, and/or a primary signalingdomain, e.g., of CD3 zeta, e.g., described herein. In one embodiment,the fusion protein is expressed by the same cell that expressed the CAR.In another embodiment, the fusion protein is expressed by a cell, e.g.,a T cell that does not express a CAR, e.g., a CD19 CAR.

In one embodiment, the extracellular domain (ECD) of a checkpointmolecule, e.g., a checkpoint molecule described herein such as, e.g.,Programmed Death 1 (PD1), can be fused to a transmembrane domain andintracellular signaling domain described herein, e.g., an intracellularsignaling domain comprising a costimulatory signaling domain such as,e.g., 41BB OX40, Cd28, CD27, and/or a primary signaling domain, e.g., ofCD3 zeta. In one embodiment, the inhibitory CAR, e.g., e.g., PD1 CAR,can be used in combination with another CAR, e.g., CD19CAR (e.g., aCD19RCAR). In one embodiment, the PD1 RCAR (or PD1 CAR) improves thepersistence of the T cell. Examples of inhibitory molecules include PD1,PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/orCEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86,B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHCclass I, MHC class II, GAL9, adenosine, and TGFR (e.g., TGFRbeta). Inone embodiment, the inhibitory molecule CAR comprises a firstpolypeptide, e.g., of an inhibitory molecule such as PD1, PD-L1, PD-L2,CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3,VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MEW class I, MEW class II,GAL9, adenosine, and TGFR (e.g., TGFRbeta), or a fragment of any ofthese (e.g., at least a portion of an extracellular domain of any ofthese), and a second polypeptide which is an intracellular signalingdomain described herein (e.g., comprising a costimulatory domain (e.g.,41BB, CD27 or CD28, e.g., as described herein) and/or a primarysignaling domain (e.g., a CD3 zeta signaling domain described herein).

In one embodiment, the inhibitory molecule CAR comprises theextracellular domain (ECD) of PD1fused to a transmembrane domain andintracellular signaling domains such as 41BB and CD3 zeta (also referredto herein as a PD1 CAR). In one embodiment, the PD1 CAR improves thepersistence of the cell CAR-expressing cell. In one embodiment, the PD1CAR comprises the extracellular domain of PD1 indicated in SEQ ID NO:44. In one embodiment, the PD1 CAR comprises, the amino acid sequence ofSEQ ID NO:40.

In one embodiment, the PD1 CAR comprises the amino acid sequenceprovided as SEQ ID NO: 41.

In one embodiment, the PD1 CAR, e.g., the PD1 CAR described herein, isencoded by a nucleic acid sequence provided as SEQ ID NO: 42, or atleast the comprises the nucleic acid sequence encoding the extracellulardomain of PD1 (provided as SEQ ID NO: 101).

In embodiments, the inhibitory extracellular domain, has at least 70,75, 80, 85, 90, 95, 96, 97, 98, or 99% identity with, or differs by nomore than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residues fromthe corresponding residues of a naturally occurring human inhibitorymolecule, e.g., a naturally occurring human primary stimulatory moleculedisclosed herein.

In an embodiment, a nucleic acid molecule that encodes a dsRNA moleculethat inhibits expression of the molecule that modulates or regulates,e.g., inhibits, T-cell function is operably linked to a promoter, e.g.,a H1- or a U6-derived promoter such that the dsRNA molecule thatinhibits expression of the molecule that modulates or regulates, e.g.,inhibits, T-cell function is expressed, e.g., is expressed within aCAR-expressing cell. See e.g., Tiscornia G., “Development of LentiviralVectors Expressing siRNA,” Chapter 3, in Gene Transfer: Delivery andExpression of DNA and RNA (eds. Friedmann and Rossi). Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA, 2007; Brummelkamp T R,et al. (2002) Science 296: 550-553; Miyagishi M, et al. (2002) Nat.Biotechnol. 19: 497-500. In an embodiment the nucleic acid molecule thatencodes a dsRNA molecule that inhibits expression of the molecule thatmodulates or regulates, e.g., inhibits, T-cell function is present onthe same vector, e.g., a lentiviral vector, that comprises a nucleicacid molecule that encodes a component, e.g., all of the components, ofthe CAR. In such an embodiment, the nucleic acid molecule that encodes adsRNA molecule that inhibits expression of the molecule that modulatesor regulates, e.g., inhibits, T-cell function is located on the vector,e.g., the lentiviral vector, 5′- or 3′- to the nucleic acid that encodesa component, e.g., all of the components, of the CAR. The nucleic acidmolecule that encodes a dsRNA molecule that inhibits expression of themolecule that modulates or regulates, e.g., inhibits, T-cell functioncan be transcribed in the same or different direction as the nucleicacid that encodes a component, e.g., all of the components, of the CAR.

In an embodiment the nucleic acid molecule that encodes a dsRNA moleculethat inhibits expression of the molecule that modulates or regulates,e.g., inhibits, T-cell function is present on a vector other than thevector that comprises a nucleic acid molecule that encodes a component,e.g., all of the components, of the CAR. In an embodiment, the nucleicacid molecule that encodes a dsRNA molecule that inhibits expression ofthe molecule that modulates or regulates, e.g., inhibits, T-cellfunction it transiently expressed within a CAR-expressing cell. In anembodiment, the nucleic acid molecule that encodes a dsRNA molecule thatinhibits expression of the molecule that modulates or regulates, e.g.,inhibits, T-cell function is stably integrated into the genome of aCAR-expressing cell. In an embodiment, the molecule that modulates orregulates, e.g., inhibits, T-cell function is PD-1.

In embodiments, the agent that enhances the activity of a CAR-expressingcell, e.g., inhibitor of an inhibitory molecule, is administered incombination with an allogeneic CAR, e.g., an allogeneic CAR describedherein (e.g., described in the Allogeneic CAR section herein).

Natural Killer Cell Receptor (NKR) CARs

In an embodiment, the CAR molecule described herein comprises one ormore components of a natural killer cell receptor (NKR), thereby formingan NKR-CAR. The NKR component can be a transmembrane domain, a hingedomain, or a cytoplasmic domain from any of the following natural killercell receptors: killer cell immunoglobulin-like receptor (KIR), e.g.,KIR2DL1, KIR2DL2/L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2,KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1/S1, KIR3DL2, KIR3DL3, KIR2DP1, andKIR3DP1; natural cytotoxicity receptor (NCR), e.g., NKp30, NKp44, NKp46;signaling lymphocyte activation molecule (SLAM) family of immune cellreceptors, e.g., CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, andCD2F-10; Fc receptor (FcR), e.g., CD16, and CD64; and Ly49 receptors,e.g., LY49A, LY49C. The NKR-CAR molecules described herein may interactwith an adaptor molecule or intracellular signaling domain, e.g., DAP12.Exemplary configurations and sequences of CAR molecules comprising NKRcomponents are described in International Publication No. WO2014/145252,the contents of which are hereby incorporated by reference.

Split CAR

In some embodiments, the CAR-expressing cell uses a split CAR. The splitCAR approach is described in more detail in publications WO2014/055442and WO2014/055657, incorporated herein by reference. Briefly, a splitCAR system comprises a cell expressing a first CAR having a firstantigen binding domain and a costimulatory domain (e.g., 41BB), and thecell also expresses a second CAR having a second antigen binding domainand an intracellular signaling domain (e.g., CD3 zeta). When the cellencounters the first antigen, the costimulatory domain is activated, andthe cell proliferates. When the cell encounters the second antigen, theintracellular signaling domain is activated and cell-killing activitybegins. Thus, the CAR-expressing cell is only fully activated in thepresence of both antigens.

Strategies for Regulating Chimeric Antigen Receptors

There are many ways CAR activities can be regulated. In someembodiments, a regulatable CAR (RCAR) where the CAR activity can becontrolled is desirable to optimize the safety and efficacy of a CARtherapy. There are many ways CAR activities can be regulated. Forexample, inducible apoptosis using, e.g., a caspase fused to adimerization domain (see, e.g., Di Stasa et al., N Engl. J. Med. 2011Nov. 3; 365(18):1673-1683), can be used as a safety switch in the CARtherapy of the instant invention. In one embodiment, the cells (e.g., Tcells or NK cells) expressing a CAR of the present invention furthercomprise an inducible apoptosis switch, wherein a human caspase (e.g.,caspase 9) or a modified version is fused to a modification of the humanFKB protein that allows conditional dimerization. In the presence of asmall molecule, such as a rapalog (e.g., AP 1903, AP20187), theinducible caspase (e.g., caspase 9) is activated and leads to the rapidapoptosis and death of the cells (e.g., T cells or NK cells) expressinga CAR of the present invention. Examples of a caspase-based inducibleapoptosis switch (or one or more aspects of such a switch) have beendescribed in, e.g., US2004040047; US20110286980; US20140255360;WO1997031899; WO2014151960; WO2014164348; WO2014197638; WO2014197638;all of which are incorporated by reference herein.

In another example, CAR-expressing cells can also express an inducibleCaspase-9 (iCaspase-9) molecule that, upon administration of a dimerizerdrug (e.g., rimiducid (also called AP1903 (Bellicum Pharmaceuticals) orAP20187 (Ariad)) leads to activation of the Caspase-9 and apoptosis ofthe cells. The iCaspase-9 molecule contains a chemical inducer ofdimerization (CID) binding domain that mediates dimerization in thepresence of a CID. This results in inducible and selective depletion ofCAR-expressing cells. In some cases, the iCaspase-9 molecule is encodedby a nucleic acid molecule separate from the CAR-encoding vector(s). Insome cases, the iCaspase-9 molecule is encoded by the same nucleic acidmolecule as the CAR-encoding vector. The iCaspase-9 can provide a safetyswitch to avoid any toxicity of CAR-expressing cells. See, e.g., Song etal. Cancer Gene Ther. 2008; 15(10):667-75; Clinical Trial Id. No.NCT02107963; and Di Stasi et al. N. Engl. J. Med. 2011; 365:1673-83.

Alternative strategies for regulating the CAR therapy of the instantinvention include utilizing small molecules or antibodies thatdeactivate or turn off CAR activity, e.g., by deleting CAR-expressingcells, e.g., by inducing antibody dependent cell-mediated cytotoxicity(ADCC).

For example, CAR-expressing cells described herein may also express anantigen that is recognized by molecules capable of inducing cell death,e.g., ADCC or complement-induced cell death. For example, CAR expressingcells described herein may also express a receptor capable of beingtargeted by an antibody or antibody fragment. Examples of such receptorsinclude EpCAM, VEGFR, integrins (e.g., integrins αvβ3, α4, αI¾β3, α4β7,α5β1, αvβ3, αv), members of the TNF receptor superfamily (e.g.,TRAIL-R1, TRAIL-R2), PDGF Receptor, interferon receptor, folatereceptor, GPNMB, ICAM-1, HLA-DR, CEA, CA-125, MUC1, TAG-72, IL-6receptor, 5T4, GD2, GD3, CD2, CD3, CD4, CD5, CD1 1, CD1 1 a/LFA-1, CD15,CD18/ITGB2, CD19, CD20, CD22, CD23/1gE Receptor, CD25, CD28, CD30, CD33,CD38, CD40, CD41, CD44, CD51, CD52, CD62L, CD74, CD80, CD125,CD147/basigin, CD152/CTLA-4, CD154/CD40L, CD195/CCR5, CD319/SLAMF7, andEGFR, and truncated versions thereof (e.g., versions preserving one ormore extracellular epitopes but lacking one or more regions within thecytoplasmic domain).

For example, a CAR-expressing cell described herein may also express atruncated epidermal growth factor receptor (EGFR) which lacks signalingcapacity but retains the epitope that is recognized by molecules capableof inducing ADCC, e.g., cetuximab (ERBITUX®), such that administrationof cetuximab induces ADCC and subsequent depletion of the CAR-expressingcells (see, e.g., WO2011/056894, and Jonnalagadda et al., Gene Ther.2013; 20(8)853-860). Another strategy includes expressing a highlycompact marker/suicide gene that combines target epitopes from both CD32and CD20 antigens in the CAR-expressing cells described herein, whichbinds rituximab, resulting in selective depletion of the CAR-expressingcells, e.g., by ADCC (see, e.g., Philip et al., Blood. 2014;124(8)1277-1287). Other methods for depleting CAR-expressing cellsdescribed herein include administration of CAMPATH, a monoclonalanti-CD52 antibody that selectively binds and targets maturelymphocytes, e.g., CAR-expressing cells, for destruction, e.g., byinducing ADCC. In other embodiments, the CAR-expressing cell can beselectively targeted using a CAR ligand, e.g., an anti-idiotypicantibody. In some embodiments, the anti-idiotypic antibody can causeeffector cell activity, e.g., ADCC or ADC activities, thereby reducingthe number of CAR-expressing cells. In other embodiments, the CARligand, e.g., the anti-idiotypic antibody, can be coupled to an agentthat induces cell killing, e.g., a toxin, thereby reducing the number ofCAR-expressing cells. Alternatively, the CAR molecules themselves can beconfigured such that the activity can be regulated, e.g., turned on andoff, as described below.

In other embodiments, a CAR-expressing cell described herein may alsoexpress a target protein recognized by the T cell depleting agent. Inone embodiment, the target protein is CD20 and the T cell depletingagent is an anti-CD20 antibody, e.g., rituximab. In such embodiment, theT cell depleting agent is administered once it is desirable to reduce oreliminate the CAR-expressing cell, e.g., to mitigate the CAR inducedtoxicity. In other embodiments, the T cell depleting agent is ananti-CD52 antibody, e.g., alemtuzumab, as described in the Examplesherein.

In other embodiments, an RCAR comprises a set of polypeptides, typicallytwo in the simplest embodiments, in which the components of a standardCAR described herein, e.g., an antigen binding domain and anintracellular signalling domain, are partitioned on separatepolypeptides or members. In some embodiments, the set of polypeptidesinclude a dimerization switch that, upon the presence of a dimerizationmolecule, can couple the polypeptides to one another, e.g., can couplean antigen binding domain to an intracellular signalling domain. In oneembodiment, a CAR of the present invention utilizes a dimerizationswitch as those described in, e.g., WO2014127261, which is incorporatedby reference herein. Additional description and exemplary configurationsof such regulatable CARs are provided herein and in, e.g., paragraphs527-551 of International Publication No. WO 2015/090229 filed Mar. 13,2015, which is incorporated by reference in its entirety. In someembodiments, an RCAR involves a switch domain, e.g., a FKBP switchdomain, as set out SEQ ID NO: 92, or comprise a fragment of FKBP havingthe ability to bind with FRB, e.g., as set out in SEQ ID NO: 93. In someembodiments, the RCAR involves a switch domain comprising a FRBsequence, e.g., as set out in SEQ ID NO: 94, or a mutant FRB sequence,e.g., as set out in any of SEQ ID Nos. 95-100.

Co-expression of CAR with a Chemokine Receptor

In embodiments, the CAR-expressing cell described herein furthercomprises a chemokine receptor molecule. Transgenic expression ofchemokine receptors CCR2b or CXCR2 in T cells enhances trafficking toCCL2- or CXCL1-secreting solid tumors including melanoma andneuroblastoma (Craddock et al., J Immunother. 2010 October; 33(8):780-8and Kershaw et al., Hum Gene Ther. 2002 Nov. 1; 13(16):1971-80). Thus,without wishing to be bound by theory, it is believed that chemokinereceptors expressed in CAR-expressing cells that recognize chemokinessecreted by tumors, e.g., solid tumors, can improve homing of theCAR-expressing cell to the tumor, facilitate the infiltration of theCAR-expressing cell to the tumor, and enhances antitumor efficacy of theCAR-expressing cell. The chemokine receptor molecule can comprise anaturally occurring or recombinant chemokine receptor or achemokine-binding fragment thereof. A chemokine receptor moleculesuitable for expression in a CAR-expressing cell described hereininclude a CXC chemokine receptor (e.g., CXCR1, CXCR2, CXCR3, CXCR4,CXCR5, CXCR6, or CXCR7), a CC chemokine receptor (e.g., CCR1, CCR2,CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, or CCR11), a CX3Cchemokine receptor (e.g., CX3CR1), a XC chemokine receptor (e.g., XCR1),or a chemokine-binding fragment thereof. In one embodiment, thechemokine receptor molecule to be expressed with a CAR described hereinis selected based on the chemokine(s) secreted by the tumor. In oneembodiment, the CAR-expressing cell described herein further comprises,e.g., expresses, a CCR2b receptor or a CXCR2 receptor. In an embodiment,the CAR described herein and the chemokine receptor molecule are on thesame vector or are on two different vectors. In embodiments where theCAR described herein and the chemokine receptor molecule are on the samevector, the CAR and the chemokine receptor molecule are each undercontrol of two different promoters or are under the control of the samepromoter.

Split CAR

In some embodiments, the CAR-expressing cell uses a split CAR. The splitCAR approach is described in more detail in publications WO2014/055442and WO2014/055657. Briefly, a split CAR system comprises a cellexpressing a first CAR having a first antigen binding domain and acostimulatory domain (e.g., 41BB), and the cell also expresses a secondCAR having a second antigen binding domain and an intracellularsignaling domain (e.g., CD3 zeta). When the cell encounters the firstantigen, the costimulatory domain is activated, and the cellproliferates. When the cell encounters the second antigen, theintracellular signaling domain is activated and cell-killing activitybegins. Thus, the CAR-expressing cell is only fully activated in thepresence of both antigens.

Pharmaceutical Compositions and Treatments

Pharmaceutical compositions may comprise a CAR-expressing cell, e.g., aplurality of CAR-expressing cells, as described herein, in combinationwith one or more pharmaceutically or physiologically acceptablecarriers, diluents or excipients. Such compositions may comprise bufferssuch as neutral buffered saline, phosphate buffered saline and the like;carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol;proteins; polypeptides or amino acids such as glycine; antioxidants;chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminumhydroxide); and preservatives. Compositions can be, e.g., formulated forintravenous administration.

Pharmaceutical compositions of the present disclosure may beadministered in a manner appropriate to the disease to be treated (orprevented). The quantity and frequency of administration will bedetermined by such factors as the condition of the patient, and the typeand severity of the patient's disease, although appropriate dosages maybe determined by clinical trials.

In one embodiment, the pharmaceutical composition is substantially freeof, e.g., there are no detectable levels of a contaminant, e.g., acontaminant described in paragraph 1009 of International ApplicationWO2015/142675, filed Mar. 13, 2015, which is herein incorporated byreference in its entirety.

When “an immunologically effective amount,” “an anti-tumor effectiveamount,” “a tumor-inhibiting effective amount,” or “therapeutic amount”is indicated, the precise amount of the compositions of the presentinvention to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising the immune effector cells (e.g., T cells, NK cells) describedherein may be administered at a dosage of 10⁴ to 10⁹ cells/kg bodyweight, in some instances 10⁵ to 10⁶ cells/kg body weight, including allinteger values within those ranges. T cell compositions may also beadministered multiple times at these dosages. The cells can beadministered by using infusion techniques that are commonly known inimmunotherapy (see, e.g., Rosenberg et al., NEW ENG. J. OF MED.319:1676, 1988).

In some embodiments, a dose of CAR cells (e.g., CD19 CAR cells)comprises about 1×10⁶, 1.1×10⁶, 2×10⁶, 3.6×10⁶, 5×10⁶, 1×10⁷, 1.8×10⁷,2×10⁷, 5×10⁷, 1×10⁸, 2×10⁸, or 5×10⁸ cells/kg. In some embodiments, adose of CAR cells (e.g., CD19 CAR cells) comprises at least about 1×10⁶,1.1×10⁶, 2×10⁶, 3.6×10⁶, 5×10⁶, 1×10⁷, 1.8×10⁷, 2×10⁷, 5×10⁷, 1×10⁸,2×10⁸, or 5×10⁸ cells/kg. In some embodiments, a dose of CAR cells(e.g., CD19 CAR cells) comprises up to about 1×10⁶, 1.1×10⁶, 2×10⁶,3.6×10⁶, 5×10⁶, 1×10⁷, 1.8×10⁷, 2×10⁷, 5×10⁷, 1×10⁸, 2×10⁸, or 5×10⁸cells/kg. In some embodiments, a dose of CAR cells (e.g., CD19 CARcells) comprises about 1.1×10⁶-1.8×10⁷ cells/kg. In some embodiments, adose of CAR cells (e.g., CD19 CAR cells) comprises about 1×10⁷, 2×10⁷,5×10⁷, 1×10⁸, 2×10⁸, 5×10⁸, 1×10⁹, 2×10⁹, or 5×10⁹ cells. In someembodiments, a dose of CAR cells (e.g., CD19 CAR cells) comprises atleast about 1×10⁷, 2×10⁷, 5×10⁷, 1×10⁸, 2×10⁸, 5×10⁸, 1×10⁹, 2×10⁹, or5×10⁹ cells. In some embodiments, a dose of CAR cells (e.g., CD19 CARcells) comprises up to about 1×10⁷, 2×10⁷, 5×10⁷, 1×10⁸, 2×10⁸, 5×10⁸,1×10⁹, 2×10⁹, or 5×10⁹ cells.

In certain aspects, it may be desired to administer activated immuneeffector cells (e.g., T cells, NK cells) to a subject and thensubsequently redraw blood (or have an apheresis performed), activateimmune effector cells (e.g., T cells, NK cells) therefrom according tothe present disclosure, and reinfuse the patient with these activatedand expanded immune effector cells (e.g., T cells, NK cells). Thisprocess can be carried out multiple times every few weeks. In certainaspects, immune effector cells (e.g., T cells, NK cells) can beactivated from blood draws of from 10 cc to 400 cc. In certain aspects,immune effector cells (e.g., T cells, NK cells) are activated from blooddraws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100cc.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, by intravenous (i.v.) injection, orintraperitoneally. In one aspect, the T cell compositions describedherein are administered to a patient by intradermal or subcutaneousinjection. In one aspect, the T cell compositions described herein areadministered by i.v. injection. The compositions of immune effectorcells (e.g., T cells, NK cells) may be injected directly into a tumor,lymph node, or site of infection.

In a particular exemplary aspect, subjects may undergo leukapheresis,wherein leukocytes are collected, enriched, or depleted ex vivo toselect and/or isolate the cells of interest, e.g., T cells. These T cellisolates may be expanded by methods known in the art and treated suchthat one or more CAR constructs described herein may be introduced,thereby creating a CAR T cell of the present disclosure. Subjects inneed thereof may subsequently undergo standard treatment with high dosechemotherapy followed by peripheral blood stem cell transplantation. Incertain aspects, following or concurrent with the transplant, subjectsreceive an infusion of the expanded CAR T cells described herein. In anadditional aspect, expanded cells are administered before or followingsurgery.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art-accepted practices. Thedose for CAMPATH, for example, will generally be in the range 1 to about100 mg for an adult patient, usually administered daily for a periodbetween 1 and 30 days. A suitable daily dose is 1 to 10 mg per dayalthough in some instances larger doses of up to 40 mg per day may beused (described in U.S. Pat. No. 6,120,766).

In one embodiment, the CAR is introduced into immune effector cells(e.g., T cells, NK cells), e.g., using in vitro transcription, and thesubject (e.g., human) receives an initial administration of CAR immuneeffector cells (e.g., T cells, NK cells) of the invention, and one ormore subsequent administrations of the CAR immune effector cells (e.g.,T cells, NK cells) of the invention, wherein the one or more subsequentadministrations are administered less than 15 days, e.g., 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previousadministration. In one embodiment, more than one administration of theCAR immune effector cells (e.g., T cells, NK cells) described herein areadministered to the subject (e.g., human) per week, e.g., 2, 3, or 4administrations of the CAR immune effector cells (e.g., T cells, NKcells) of the invention are administered per week. In one embodiment,the subject (e.g., human subject) receives more than one administrationof the CAR immune effector cells (e.g., T cells, NK cells) per week(e.g., 2, 3 or 4 administrations per week) (also referred to herein as acycle), followed by a week of no CAR immune effector cells (e.g., Tcells, NK cells) administrations, and then one or more additionaladministration of the CAR immune effector cells (e.g., T cells, NKcells) (e.g., more than one administration of the CAR immune effectorcells (e.g., T cells, NK cells) per week) is administered to thesubject. In another embodiment, the subject (e.g., human subject)receives more than one cycle of CAR immune effector cells (e.g., Tcells, NK cells), and the time between each cycle is less than 10, 9, 8,7, 6, 5, 4, or 3 days. In one embodiment, the CAR immune effector cells(e.g., T cells, NK cells) are administered every other day for 3administrations per week. In one embodiment, the CAR immune effectorcells (e.g., T cells, NK cells) described herein are administered for atleast two, three, four, five, six, seven, eight or more weeks.

In one aspect, CAR-expressing cells (e.g., T cells, NK cells) asdescribed herein such as, e.g., CD19 CAR-expressing cells, e.g., CTL019are generated using lentiviral viral vectors, such as lentivirus.CAR-expressing cells generated that way can have stable CAR expression.

In one aspect, CAR-expressing cells (e.g., T cells, NK cells)transiently express CAR vectors for 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15 days after transduction. Transient expression of CARs can beeffected by RNA CAR vector delivery. In one aspect, the CAR RNA istransduced into the cell by electroporation.

A potential issue that can arise in patients being treated usingtransiently expressing CAR cells, e.g., T cells (particularly withmurine scFv bearing CAR-expressing cells (e.g., T cells, NK cells)) isanaphylaxis after multiple treatments. Without being bound by thistheory, it is believed that such an anaphylactic response might becaused by a patient developing humoral anti-CAR response, i.e., anti-CARantibodies having an anti-IgE isotype. It is thought that a patient'santibody producing cells undergo a class switch from IgG isotype (thatdoes not cause anaphylaxis) to IgE isotype when there is a ten tofourteen day break in exposure to antigen.

If a patient is at high risk of generating an anti-CAR antibody responseduring the course of transient CAR therapy (such as those generated byRNA transductions), CAR-expressing cell (e.g., T cell, NK cell) infusionbreaks should not last more than ten to fourteen days.

In some embodiments of any of the aforesaid methods, the method furtherincludes administering one or more doses of a cell (e.g., an immune cellcontaining a CAR nucleic acid or CAR polypeptide as described herein),to a mammal (e.g., a mammal having a cancer, e.g., a mammal that is oris identified as being a responder, complete responder, partialresponder, non-responder, relapser, or non-relapser according to themethods herein). In some embodiments, the one or more doses of CAR cells(e.g., CD19 CAR cells) comprises at least about 1×10⁶, 5×10⁶, 1×10⁷,2×10⁷, 5×10⁷, 1×10⁸, 2×10⁸, 5×10⁸, 1×10⁹, 2×10⁹, or 5×10⁹ cells.

In one embodiment, up to 10, 9, 8, 7, 6, 5, 4, 3, or 2 doses of cellsare administered. In other embodiments, one, two, three, four, five or 6doses of the cells are administered to the mammal, e.g., in a treatmentinterval of one, two, three, four or more weeks. In one embodiment, upto 6 doses are administered in two weeks. The doses may the same ordifferent. In one embodiment, a lower dose is administered initially,followed by one or more higher doses. In one exemplary embodiment, thelower dose is about 1×10⁵ to 1×10⁹ cells/kg, or 1×10⁶ to 1×10⁸ cells/kg;and the higher dose is about 2×10⁵ to 2×10⁹ cells/kg or 2×10⁶ to 2×10⁸cells/kg, followed by 3-6 doses of about 4×10⁵ to 4×10⁹ cells/kg, or4×10⁶ to 4×10⁸ cells/kg.

In one embodiment, the one or more doses of the cells are administeredafter one or more lymphodepleting therapies, e.g., a lymphodepletingchemotherapy. In one embodiment, the lymphodepleting therapy includes achemotherapy (e.g., cyclophosphamide).

In one embodiment, the one or more doses is followed by a celltransplant, e.g., an allogeneic hematopoietic stem cell transplant. Forexample, the allogeneic hematopoietic stem cell transplant occursbetween about 20 to about 35 days, e.g., between about 23 and 33 days.

Biopolymer Delivery Methods

In some embodiments, one or more CAR-expressing cells as disclosedherein can be administered or delivered to the subject via a biopolymerscaffold, e.g., a biopolymer implant. Biopolymer scaffolds can supportor enhance the delivery, expansion, and/or dispersion of theCAR-expressing cells described herein. A biopolymer scaffold comprises abiocompatible (e.g., does not substantially induce an inflammatory orimmune response) and/or a biodegradable polymer that can be naturallyoccurring or synthetic. Exemplary biopolymers are described, e.g., inparagraphs 1004-1006 of International Application WO2015/142675, filedMar. 13, 2015, which is herein incorporated by reference in itsentirety.

Exemplary Computer System

Various computer systems can be specially configured to leverageinformation returned on potential cancer responder status indicators(for example, ALL and CLL responder status indicator as described hereinsuch as, e.g., complete responder, partial responder, non-responder). Insome embodiments, the computer system can determine and presentinformation on confidence levels associated with various bio-markersand/or indicators for cancer (e.g. a hematological cancer) such as ALLand CLL. For example, the computer systems can evaluate whether a testconducted on a subject indicates a gene signature of a completeresponder, partial responder or non-responder, along with a degree ofconfidence associated with the subject responder classification. Infurther examples, the system can provide an indication and/orrecommendation on increasing the degree of confidence associated withthe predicted responder classification. For example, the system can beconfigured to evaluate any tests and tested biomarkers and/or indicatorsof cancer that have been performed for a subject against anothercharacteristic identified as independent and/or additive of the existingdata. In an embodiment, the system can be configured to evaluate anytests and tested biomarkers and/or cancer indicators (e.g., ahematological cancer such as CLL and ALL) that have been performed for asubject against another characteristic identified as independent and/oradditive of the existing data. The system can determine when anadditional biomarker and/or indicator (e.g., gene signature) wouldincrease confidence associated with, for example, a change in responderclassification. The system can recommend testing of any identifiedcharacteristic accordingly.

In some embodiments, an interactive system for identification,assessment and/or treatment of a subject having cancer (e.g., ahematological cancer such as ALL and CLL) can be provided. In anembodiment, an interactive system for identification, assessment and/ortreatment of a subject having cancer (e.g., a hematological cancer suchas ALL and CLL) can be provided. According to one embodiment, the systemcan be configured to accept user input regarding degree of confidence ofa subject assessment. Responsive to the user entered degree ofconfidence, the system can determine test characteristics to include inan evaluation model. In one example, the system includes specificationof independent indicators for disease activity in a subject (e.g.,patient) population. The system can be configured to estimate a degreeof confidence in a determination of disease activity or a prediction offuture disease activity based on what independent indicators are used.The system can be further configured to determined and/or recommendvarious combinations of the determined independent indicators to improvea degree of confidence in an evaluation.

According to another aspect, a computer system can be speciallyconfigured to evaluate indicators for cancer (e.g., a hematologicalcancer such as ALL and CLL). In an embodiment, a computer system can bespecially configured to evaluate indicators for ALL and/or CLL. Thesystem can be configured to generate a multivariate model, wherein themultivariate model excludes correlated indicators. In some examples, thesystem can be configured to identify correlated indicators responsive toevaluating returned test results within a subject (e.g., patient)population having one or more of the indicators. For example, the systemcan execute regression model analysis to control for various parameters,including, for example, subject age, race, sex, and the presence ofother indicators. Responsive to eliminating correlated indicators, thesystem can generate a model of one or more independent indicators. Insome embodiments, the system can be configured to select variouscombinations of the one or more independent indicators and can furtheraccess evaluations (including, for example, evaluating the combinationdirectly) to present information on a confidence level associated withrespective selections. The system selected models can be used togenerate an expected change in disease activity with the determinedconfidence level.

In an embodiment, the disclosure provides a system for evaluating cancer(e.g., a hematological cancer such as ALL and CLL) in a subject,comprising:

at least one processor operatively connected to a memory, the at leastone processor when executing is configured to:

acquire a value of responder status that comprises a measure of a CD19CAR-expressing cell (e.g., T cell, NK cell) gene set signature and acombination of one or more of:

a biomarker listed in Table 1A, Table 1B, Table 7A, Table 7B, Table 8,Table 9, Table 10, Table 14, Table 15, Table 16 (e.g., CCL20, IL-17aand/or IL-6), Table 17, Table 18, Table 20, a CD27 biomarker, a CD45RObiomarker, a PD-1 biomarker, a LAG-3 biomarker, a TIM-3 biomarker, anIL2RA biomarker, an IL21 biomarker, a CD4 biomarker, a CD8 biomarker, aTH1+ helper T cell gene set signature, a TH2+ helper T cell gene setsignature, and a memory T cell (e.g., a CD8+ memory T cell, e.g., anaïve T cell (T_(N)), e.g. a memory stem cell (T_(SCM)), e.g. a centralmemory T cell (T_(CM)), e.g. an effector memory T cell (T_(EM))) geneset signature; and

responsive to a determination of the value of responder status, performone, two, three, four or more of:

identify the subject as a complete responder, partial responder, ornon-responder;

recommend a CAR-expressing cell (e.g., T cell, NK cell) therapy (e.g., aCD19 CAR-expressing cell (e.g., T cell, NK cell) therapy as describedherein, such as, e.g., CTL019);

recommend a selection or alteration of a dosing of a CAR-expressing cell(e.g., T cell, NK cell) therapy; or

an alternative therapy, e.g., a standard of care for the particularcancer.

In an embodiment, the invention provides a system for evaluating cancer(e.g., a hematological cancer such as ALL and CLL) in a subject,comprising: at least one processor operatively connected to a memory,the at least one processor when executing is configured to: acquire avalue of responder status that comprises a measure of a CD19CAR-expressing cell (e.g., T cell, NK cell) gene set signature and acombination of one or more of: a biomarker listed in Table 1A, Table 1B,Table 7A, Table 7B, Table 8, Table 9, Table 10, Table 14, Table 15,Table 16 (e.g., CCL20, IL-17a and/or IL-6), Table 17, Table 18, Table20, PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L, and KLRG1; andresponsive to a determination of the value of responder status, performone, two, three, four or more of: identify the subject as a completeresponder, partial responder, or non-responder; recommend aCAR-expressing cell therapy (e.g., a CD19 CAR-expressing cell therapy asdescribed herein, such as, e.g., CTL019); recommend a selection oralteration of a dosing of a CAR-expressing cell therapy; or analternative therapy.

FIG. 16 is a block diagram of a distributed computer system 200, inwhich various aspects and functions in accord with the presentdisclosure may be practiced. The distributed computer system 200 mayinclude one or more computer systems. For example, as illustrated, thedistributed computer system 200 includes three computer systems 202, 204and 206. As shown, the computer systems 202, 204 and 206 areinterconnected by, and may exchange data through, a communicationnetwork 208. The network 208 may include any communication networkthrough which computer systems may exchange data. To exchange data viathe network 208, the computer systems 202, 204, and 206 and the network208 may use various methods, protocols and standards including, amongothers, token ring, Ethernet, Wireless Ethernet, Bluetooth, radiosignaling, infra-red signaling, TCP/IP, UDP, HTTP, FTP, SNMP, SMS, MMS,SS2, JSON, XML, REST, SOAP, CORBA IIOP, RMI, DCOM and Web Services.

According to some embodiments, the functions and operations discussedfor identifying, treating or preventing cancer (e.g., a hematologicalcancer such as ALL and CLL) in a subject can be executed on computersystems 202, 204 and 206 individually and/or in combination. Forexample, the computer systems 202, 204, and 206 support, for example,participation in a collaborative operations, which may include analyzingtreatment data captured on a patient population. In one alternative, asingle computer system (e.g., 202) can analyze treatment data capturedon a subject (e.g., patient) population to develop characterizationmodels and/or identify independent indicators for disease activity. Thecomputer systems 202, 204 and 206 may include personal computing devicessuch as cellular telephones, smart phones, tablets, etc., and may alsoinclude desktop computers, laptop computers, etc.

Various aspects and functions in accord with the present disclosure maybe implemented as specialized hardware or software executing in one ormore computer systems including the computer system 202 shown in FIG.16. In one embodiment, computer system 202 is a computing devicespecially configured to execute the processes and/or operationsdiscussed above. For example, the system can present user interfaces toend-users that present treatment information, diagnostic information,and confidence levels associated with biomarkers and/or geneticindicators, among other options. As depicted, the computer system 202includes at least one processor 210 (e.g., a single core or a multi-coreprocessor), a memory 212, a bus 214, input/output interfaces (e.g., 216)and storage 218. The processor 210, may include one or moremicroprocessors or other types of controllers, and can perform a seriesof instructions that manipulate data (e.g., treatment data, testingdata, etc.). As shown, the processor 210 is connected to other systemcomponents, including a memory 212, by an interconnection element (e.g.,the bus 214).

The memory 212 and/or storage 218 may be used for storing programs anddata during operation of the computer system 202. For example, thememory 212 may be a relatively high performance, volatile, random accessmemory such as a dynamic random access memory (DRAM) or static memory(SRAM). In addition, the memory 212 may include any device for storingdata, such as a disk drive or other non-volatile storage device, such asflash memory, solid state, or phase-change memory (PCM). In furtherembodiments, the functions and operations discussed with respect toidentifying, treating or preventing cancer (e.g., ALL and/or CLL) in asubject can be embodied in an application that is executed on thecomputer system 202 from the memory 212 and/or the storage 218.

Computer system 202 also includes one or more interfaces 216 such asinput devices, output devices, and combination input/output devices. Theinterfaces 216 may receive input, provide output, or both. The storage218 may include a computer-readable and computer-writeable nonvolatilestorage medium in which instructions are stored that define a program tobe executed by the processor. The storage system 218 also may includeinformation that is recorded, on or in, the medium, and this informationmay be processed by the application. A medium that can be used withvarious embodiments may include, for example, optical disk, magneticdisk or flash memory, SSD, among others.

Further, the invention is not limited to a particular memory system orstorage system. Although the computer system 202 is shown by way ofexample as one type of computer system upon which various functions foridentifying, treating or preventing cancer (e.g., a hematological cancersuch as ALL and CLL) in a subject may be practiced, aspects of theinvention are not limited to being implemented on the computer system,shown in FIG. 16. Various aspects and functions in accord with thepresent invention may be practiced on one or more computers havingdifferent architectures or components than that shown in FIG. 16.

In some embodiments, the computer system 202 may include an operatingsystem that manages at least a portion of the hardware components (e.g.,input/output devices, touch screens, cameras, etc.) included in computersystem 202. One or more processors or controllers, such as processor210, may execute an operating system which may be, among others, aWindows-based operating system (e.g., Windows NT, ME, XP, Vista, 2, 8,or RT) available from the Microsoft Corporation, an operating systemavailable from Apple Computer (e.g., MAC OS, including System X), one ofmany Linux-based operating system distributions (for example, theEnterprise Linux operating system available from Red Hat Inc.), aSolaris operating system available from Sun Microsystems, or a UNIXoperating systems available from various sources. Many other operatingsystems may be used, including operating systems designed for personalcomputing devices (e.g., iOS, Android, etc.) and embodiments are notlimited to any particular operating system.

According to one embodiment, the processor and operating system togetherdefine a computing platform on which applications may be executed.Additionally, various functions for identifying, treating or preventingcancer (e.g., a hematological cancer such as ALL and CLL) in a subjectmay be implemented in a non-programmed environment (for example,documents created in HTML, XML or other format that, when viewed in awindow of a browser program, render aspects of a graphical-userinterface or perform other functions). Further, various embodiments inaccord with aspects of the present disclosure may be implemented asprogrammed or non-programmed components, or any combination thereof.Thus, the disclosure is not limited to a specific programming languageand any suitable programming language could also be used.

EXEMPLIFICATION

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples specifically point out various aspects of the presentinvention, and are not to be construed as limiting in any way theremainder of the disclosure.

Example 1: Identification of Novel Transcriptional Gene Signatures thatPredict Subject Response to CD19 CAR-Expressing Cell Therapy in ChronicLymphoid Leukemia (CLL) and Acute Lymphoblastic Leukemia (ALL) UsingWhole Genome RNAseq and Unbiased Feature Selection

The present Example describes the identification of noveltranscriptional gene signatures that predict patient response to CD19CAR-expressing cell (e.g., T cell, NK cell) therapy (e.g., CTL019therapy) in Chronic Lymphoid Leukemia (CLL) and Acute LymphoblasticLeukemia (ALL), for use in accordance with the present invention.

Among other things, the present Example describes novel gene signaturesbased on mRNA expression levels of selected genes in apheresis andmanufactured CD19 CAR-expressing cell (e.g., T cell, NK cell) productsamples (e.g., CTL019) prior to re-infusion.

In particular, the present Example describes methods of unbiased featureselection to discover novel gene signatures that predict patientresponse to CD19 CAR-expressing cell (e.g., T cell, NK cell) therapy(e.g., CTL019) in CLL and ALL, for use in accordance with the presentinvention.

Novel gene signatures based on mRNA expression levels in apheresis andmanufactured CD19 CAR-expressing cell product samples prior tore-infusion have been identified that predict patient response to CD19CAR-expressing cell therapy in Chronic Lymphoid Leukemia (CLL) and AcuteLymphoblastic Leukemia (ALL). The identified signatures were discoveredin a whole genome RNAseq study of manufactured product samples whichincluded 7 ALL subject samples and 21 CLL subject samples. ALL subjectsamples (7 total) were taken from subjects (e.g., patients) withcomplete response to CD19 CAR-expressing cell therapy. CLL subjectsamples (21 total) were stratified as follows: biological samples weretaken from 2 patients that were complete responders (CRs) to CTL019therapy, 6 patients that were partial responders (PRs), and 13non-responders (NRs). The gene signatures were then investigated in asubset of the above patients where samples were collected at apheresis.Several gene signatures discriminating responders from non-responders inmanufactured product and apheresis samples were discovered and aredescribed further in Example 2. Healthy donor samples with manufacturedproduct (i.e., reference samples) were acquired and used as a referencelevel.

Novel gene signatures were then discovered using various data analyticalapproaches: 1) unbiased feature selection; 2) gene set analysis; and 3)differential expression analysis of selected genes of interest. Gene setanalysis (2) and differential expression analysis (3) are discussed infurther detail in Example 2.

Novel gene signatures derived from unbiased feature selection werediscovered by determining which genes were differentially expressedbetween the CRs and NRs and between CRs and PRs. Genes were defined asdifferentially expressed if their differential expression wasstatistically significant with a FDR p-value cutoff of 0.1. The genelists for the CR vs NR comparison (N=128) and CR vs PR comparison (N=34)are tabulated in Table 1A-B.

Without wishing to be bound by a particular theory, these data indicatethat the differentiation state of T cells in apheresis or CD19CAR-expressing cell (e.g., T cell, NK cell) product (e.g., CTL019)correlate with subject response (i.e., CR, PR, or NR). Gene signaturesfor T cells from CR are in a more unstimulated/undifferentiated state.In addition, memory T cell subsets are differentially enriched betweenCRs versus NRs, with CRs showing similarity to naive T cells (T_(N)) andT memory stem cells (T_(SCM)). An exemplary schematic illustrating theprogression of a naïve T cell (T_(N)) through the memory T cell subsetstages, into an effector memory T cell (T_(EM)) is shown in FIG. 3).

Complete responders to CTL019 therapy have a significantly higher % ofCD8+ T cells that express the co-stimulatory molecule CD27 but lack theantigen-experienced T cell marker CD45RO compared to the non-responders.In an embodiment, the threshold for this discrimination was 7%CD27+CD45RO− cells in the CD8+ population. In an embodiment, a completeresponder is defined as 7% or greater CD27+CD45RO− cells in the CD8+population. Without wishing to be bound by a particular theory, thestate of memory T cells in CTL019 samples is likely a major component ofresponse (FIG. 3).

These data demonstrate that CRs are more like resting T_(EFF) cells,resting T_(REG) cells, naïve CD4 cells, unstimulated memory cells andearly memory T cells, whereas NRs are more like activated T_(EFF) cells,activated T_(REG) cells, activated TH1 and TH2 cells, stimulated memorycells, and late T memory cells.

TABLE 1A Table 1A: Comparison of Complete Responders (CR) vs.Non-responders (NR) Gene Unigene Accession No. FDR C16orf74 Hs.461655NM_206967 6.47E−05 uc021oxm 1.67E−03 uc021ygq 2.83E−03 uc021oxp 3.31E−03SNED1 Hs.471834 NM_001080437 9.55E−03 ADAM19 Hs.483944 NM_0332741.34E−02 FAIM2 Hs.567424 NM_012306 1.82E−02 WHAMMP2 1.82E−02 LOC730091Hs.659905 1.84E−02 TCF7 Hs.573153 NM_201633, NM_201632, 1.84E−02NM_001134851, NM_001134852. NM_213648, NM_003202, NM_201634 TTLL2Hs.520554 NM_031949 1.84E−02 LY9 Hs.403857 NM_002348, NM_0010336672.03E−02 uc021tnc 2.13E−02 TRIL Hs.21572 NM_014817 2.28E−02 uc004crn2.28E−02 DPEP2 Hs.372633 NM_022355 2.28E−02 GZMB Hs.1051 NM_0041312.35E−02 FAM102A Hs.568044 NM_203305, NM_001035254 2.35E−02 ALS2CLHs.517937 NM_147129 2.35E−02 EPHA4 Hs.371218 NM_004438 2.35E−02 IKBIPHs.252543 NM_201612, NM_201613, 2.88E−02 NM_153687 HBEGF Hs.799NM_001945 2.88E−02 LHFPL3 Hs.659164 NM_199000 2.88E−02 RCAN2 Hs.440168NM_005822 2.97E−02 MFGE8 Hs.3745 NM_005928, NM_001114614 2.97E−02 IL24Hs.723317, NM_006850, NM_181339 2.97E−02 Hs.58831 FAIM3 Hs.723317,NM_001142472, 2.97E−02 Hs.58831 NM_001142473, NM_005449 CDKN1B Hs.238990NM_004064 2.99E−02 AQP3 Hs.234642 NM_004925 3.06E−02 GPR155 Hs.516604NM_001033045, NM_152529 3.06E−02 HS6ST2 Hs.385956 NM_147175,NM_001077188 3.25E−02 SNORD85 3.25E−02 uc022cci 3.25E−02 GSTM1 Hs.301961NM_000561, NM_146421 3.27E−02 VSIG1 Hs.177164 NM_001170553, NM_1826073.86E−02 VIPR1 Hs.348500 NM_004624 3.86E−02 RCAN3 Hs.656799 NM_0134414.45E−02 ADHFE1 Hs.720023 NM_144650 4.49E−02 HSPH1 Hs.36927 NM_0066444.62E−02 ENPP6 Hs.297814 NM_153343 4.74E−02 RORC Hs.607993, NM_005060,NM_001001523 4.83E−02 Hs.256022 TRIB2 Hs.627749, NM_021643 4.96E−02Hs.467751 LRP8 Hs.576154 NM_001018054, NM_004631, 4.96E−02 NM_033300,NM_017522 RGS17 Hs.166313 NM_012419 5.05E−02 TAAR3 Hs.679662 5.18E−02C5orf41 Hs.484195 NM_153607, 5.27E−02 NM_001168394, NM_001168393 MIR31835.27E−02 LTA Hs.36 NM_001159740, NM_000595 5.27E−02 KLHL24 Hs.407709NM_017644 5.28E−02 PIK3IP1 Hs.26670 NM_001135911, NM_052880 5.28E−02MAP3K1 Hs.653654 NM_005921 5.29E−02 VWC2L Hs.534834 NM_0010805005.29E−02 IDI2-AS1 5.29E−02 DUSP4 Hs.417962 NM_001394, NM_057158 5.29E−02SKIL Hs.581632 NM_001145098, 5.77E−02 NM_001145097, NM_005414 uc021oxf5.86E−02 AMICA1 Hs.16291 NM_153206, NM_001098526 5.86E−02 TP53INP1Hs.492261 NM_001135733, NM_033285 5.86E−02 GDAP1L1 Hs.517059 NM_0240346.00E−02 HK2 Hs.591588, NM_000189 6.43E−02 Hs.406266 CBLL1 Hs.592271NM_024814 6.44E−02 PSD3 Hs.434255 NM_015310, NM_206909 6.44E−02 PUS7Hs.520619 NM_019042 6.44E−02 MSMO1 6.63E−02 IDI1 Hs.283652 NM_0045086.63E−02 HRH4 Hs.287388 NM_001160166, NM_021624, 6.73E−02 NM_001143828FAM19A1 Hs.655061 NM_213609 6.73E−02 EHD4 Hs.143703 NM_139265 6.73E−02PVR Hs.171844 NM_001135768, NM_006505, 6.74E−02 NM_001135770,NM_001135769 MIR1293 6.74E−02 WDR64 Hs.723441 NM_144625 6.74E−02 CDKN1AHs.370771 NM_078467, NM_000389 6.74E−02 CACNA1I Hs.125116 NM_021096,NM_001003406 6.75E−02 C21orf63 Hs.208358 NM_058187 6.75E−02 FLJ41649Hs.654837 9.72E−02 MPP7 Hs.499159 NM_173496 9.82E−02 POP1 Hs.252828NM_001145861, 9.82E−02 NM_001145860, NM_015029 CALCOCO1 Hs.156667NM_020898, NM_001143682 9.82E−02 COL5A3 Hs.235368 NM_015719 9.82E−02LHFP Hs.507798 NM_005780 9.82E−02 CTSO Hs.75262 NM_001334 9.82E−02 LEF1Hs.555947 NM_001166119, 9.82E−02 NM_001130713, NM_001130714, NM_016269RNASET2 Hs.720966, NM_003730 9.89E−02 Hs.529989

TABLE 1B Table 1B: Comparison of Complete responders (CR) vs. Partialresponders (PR) Gene Unigene Acession No. FDR uc021oxm 0.000511026uc021oxp 0.000511026 SPTB Hs.417303 NM_000347, NM_001024858 0.019052239ALS2CL Hs.517937 NM_147129, NM_182775 0.025286191 TCF7 Hs.573153NM_201633, NM_201632, 0.025286191 NM_001134851, NM_001134852, NM_213648,NM_003202, NM_201634 TRIL Hs.21572 NM_014817 0.025286191 WDR86 Hs.647083NM_198285 0.025286191 ACSM2B Hs.567879, Hs.298252 NM_182617,NM_001105069 0.044953527 DUSP4 Hs.417962 NM_001394, NM_0571580.044953527 EFHC1 Hs.403171 NM_018100 0.044953527 HS6ST2 Hs.385956NM_147175, NM_001077188 0.044953527 TRIB2 Hs.627749, Hs.467751 NM_0216430.047492472 SQLE Hs.71465 NM_003129 0.053797924 PRR5-ARHGAP8 Hs.720401,Hs.102336 NM_001017530, NM_181333, 0.053797924 NM_181334, NM_181335,NM_015366, NM_001017526, NM_001017529, NM_001017528 C16orf74 Hs.461655NM_206967 0.056452084 TMIE Hs.185777 NM_147196 0.056452084 LOC100131176Hs.659231 0.056452084 VSIG1 Hs.177164 NM_001170553, NM_1826070.056452084 MIR3194 0.056452084 RAP1GAP2 Hs.499659 NM_015085,NM_001100398 0.057016164 FLJ13197 Hs.29725 0.084544923 TSPEAR0.084544923 uc021zdn 0.084544923 RASA3 Hs.593075 NM_007368 0.084544923OLIG3 Hs.195398 NM_175747 0.084544923 GPR155 Hs.516604 NM_001033045,NM_152529 0.084544923 uc021ygq 0.084544923 FAM19A1 Hs.655061 NM_2136090.084544923 LY9 Hs.403857 NM_002348, NM_001033667 0.084544923 ANKRD20A5P0.084544923 C21orf15 Hs.580910 0.08962672 ADHFE1 Hs.720023 NM_1446500.08962672 MIR1293 0.098706653 LOC730091 Hs.659905 0.098706653

Example 2: Identification of Novel Transcriptional Gene Signatures whichPredict Subject Response to CD19 CAR-Expressing Cell Therapy in ChronicLymphoid Leukemia (CLL) and Acute Lymphoblastic Leukemia (ALL) UsingGene Set Analysis and Differential Expression Analysis

The present Example describes the identification of noveltranscriptional gene signatures that predict patient response to CD19CAR-expressing cell (e.g., T cell, NK cell) therapy (e.g., CTL019) inCLL and ALL, for use in accordance with the present invention.

In particular, the present Example describes methods of Gene SetAnalysis to discover novel gene signatures, for use in accordance withthe present invention.

Among other things, the present Example describes novel gene signaturesbased on Gene Set Analysis, that are predictive of patent response toCD19 CAR-expressing cell (e.g., T cell, NK cell) therapy (e.g., CTL019).Gene set analysis was performed on gene sets described in Example 1, andwith gene sets from three additional datasets. FIG. 2A depicts anexemplary histogram comparing the number of samples analyzed in a wholegenome CTL019 RNAseq analysis. p=product; a=apheresis. Gene sets weresourced from (1) additional experiments were based on gene sets by Szaboet al. (described herein); (2) gene sets published by Abbas et al. inGENOME RESEARCH 2005; and (3) gene sets published by Gattinoni et al. inNATURE MEDICINE 2011. Each of these gene sets are described in furtherdetail below.

Szabo et al. gene sets that were used in the Gene Set Analysis areprovided in Table 2. Human CD4+ T cells were purified from PBMCs (5normal donors, males, ages 18-28, with no known allergies orinfections). CD4+CD25+(T reg) and CD4+CD25− (T effector) T cells wereisolated and anti-CD3/CD28 stimulated for 0 or 16 hours to yield 4conditions: (1) T reg 0 hours; (2) T reg 16 hours; (3) T effector 0hours; and (4) T effector 16 hours. Two gene sets were derived for eachcondition: one set of genes whose expression levels were up-regulated inthat condition relative to all others and another set of genes whoseexpression levels were downregulated in that condition relative to allothers. The number of genes in each gene set was determined by a foldchange cutoff (see Table 2).

TABLE 2 Table 2: Gene sets comprised of genes up or down regulated inT_(REG) and T_(EFF) at resting and activation (Szabo data set) GeneNumber of Genes Downregulated T_(REG) vs T_(EFF) 0 h (FC3 p < 0.05) 120Downregulated T_(REG) vs T_(EFF) 16 h (FC3 p < 0.05) 139 DownregulatedT_(EFF) 16 h vs 0 h (FC7 p < 0.05) 246 Upregulated T_(REG) vs T_(EFF) 16h (FC4 p < 0.05) 254 Upregulated T_(REG) vs T_(EFF) 0 h (FC4 p < 0.05)135 Upregulated T_(EFF) 16 h vs 0 h (FC9 p < 0.05) 347 UpregulatedT_(REG) 16 h vs 0 h (FC8 p < 0.05) 226

Exemplary genes according to Table 2 downregulated in Treg compared withTeff at 0 h include ABCB1, ACSL6, ADAMTS10, ADD2, AIF1, AIF1, AIF1,AIF1, AK5, AKR1E2, ALS2CL, ANK3, ANKRD55, APBA2, AREG, ATHL1, AXIN2,B4GALNT4, BACH2, BCL7A, BEND5, BHLHE40, BPGM, C10orf47, C16orf54,C1orf228, C2orf89, CA6, CACHD1, CACNA1I, CCL5, CELA1, CHD7, CHI3L2,COL18A1, COL6A1, CR2, CYB561, CYSLTR1, D4S234E, DACT1, DENND5A, DHRS3,DLG4, DLL1, DPYSL4, DSC1, EDAR, EMR1, EMR4P, ENC1, EPHA1, FCGBP, FHIT,GADD45G, GIPC3, GIPC3, GPR125, GPR160, H1F0, HDGFRP3, HIPK2, IFITM5,KCNQ1, KLF5, KLHL29, KRT72, KRT73, LASS6, LRRC24, MAN1C1, ME3, MMP28,MTUS1, NBL1, NELL2, NEO1, NKG7, NLRP6, NME4, NOG, NOSIP, NPAS2, NRCAM,OBSCN, OSBPL5, PCSK5, PDZD4, PECAM1, PLLP, PLXDC1, PPFIBP2, PRKAR1B,PTK2, RHOB, RMRP, RNF157, SATB1, SCML4, SDK2, SEC14L2, SEC14L2, SLC15A3,SLC22A17, SLC22A23, SLC40A1, SNTB1, SORBS3, SOX8, ST6GALNAC1, TCF7,TCF7, THEMIS, TMIGD2, VIPR1, WNT10B, WNT7A, ZNF467, ZNF516, and ZNF609.

Exemplary genes according to Table 2 downregulated in Treg compared toTeff 16 h include IL2, TNFSF8, NELL2, GOS2, IRF8, IFNG, IGFBP4, GPR125,CD200, GPR81, ADD2, IL21, SNORD86, TMCC2, C1orf228, SLC15A3, IL22,LRRN3, GPR171, FASLG, GZMH, NHS, MCOLN2, BACH2, TAGAP, MPZL2, PRAGMIN,DACT1, CXCL10, SLAMF6, PHGDH, CSF2, PRSS23, UHRF1, PLAC8, ISM1, BTLA,CDC20, GFOD1, HSD11B1, ME3, ZNF704, DHRS3, CXCL13, CCND1, NBL1, CRTAM,MAP6D1, H1F0, CDT1, CCL4, LIF, CD84, TRAT1, MIR155, SLAMF7, AIF1, AIF1,AIF1, AIF1, PRG4, VWCE, CHEK1, SH2D4A, MCM10, RHOU, NPAS2, NFIX, STAP1,DTL, C16orf59, CSDA, GINS2, FAM117B, ABCB1, CLC, PHEX, GDF10, RAB13,BCL7A, MAMLD1, SHF, LPIN2, AHI1, CCND3, HDGFRP3, MIR155HG, PVR, CDCA5,RRAS2, SIPA1L2, RASL10B, GAL, SNORD88C, SNORD18B, CDC6, SRD5A3, ORC6L,B3GNT5, ANK3, MCM2, MIR25, RHOBTB3, TNF, TERT, CSDAP1, CCDC64, CDC25A,ZNF367, MCM7, CASP10, LTA, MCM4, AFF3, FMNL2, TNFRSF21, AXIN2, CHD7,FABP5, XRCC2, CGREF1, CCL4L1, CCL4L2, B4GALNT4, DSCC1, CD97, PTPRK,RAD54L, EPB41L3, MYO1B, ORC1L, CHML, ZWINT, MAD2L1, NDST1, C11orfK,BEGAIN, CD55, and FABP5L3.

Exemplary genes according to Table 2 downregulated in Teff at 16 h vs 0h include ABCA7, ABCG1, ABTB1, ACCS, ADAMTS10, ADD3, AK5, ALS2CL, AMT,ANKRD55, ANXA1, AQP3, AREG, ARL4C, ARRDC2, ARRDC3, BBC3, BCL9L, BIN2,BNIP3L, BTG1, BTN3A1, C10orf110, C11orf21, C11orf35, C14orf181,C16orf54, C16orf74, C17orf108, C1orf162, C1QTNF6, C20orf111, C20orf112,C5orf39, C5orf41, C5orf41, CACNA1I, CAPS, CBX4, CCNL1, CDC14A, CDC42BPG,CECR1, CFP, CHI3L2, CITED4, CLK1, CRIP2, CSGALNACT1, CTSF, CTSW, CXCR4,CYTH4, DCHS1, DDIT3, DDX60L, DISC1, DISC1, DISC1, DISC1, DPEP2, DPYD,DUSP1, DUSP8, EDAR, EMR4P, EPHA4, EPHX1, EPHX2, ERMN, ERP27, EVI2B,FAM13A, FAM13AOS, FAM46C, FAM65B, FBXO32, FHIT, FLT3LG, FOS, FOSB,FRAT1, FYB, GABARAPL1, GABARAPL3, GADD45B, GOLGA7B, GPA33, GPRASP1,GRASP, GSTM2, GZMA, GZMK, HBP1, HERPUD2, HIST1H1C, HIST1H3A, HPCAL4,HSD17B11, ID1, IDUA, IER2, IFI44, IL10RA, IL11RA, IRF2BP2, IRS2, ITGA6,JMY, JUN, JUNB, JUND, KCNQ1, KIAA1370, KIAA1683, KLF2, KLF3, KLF4, KLF5,KLF6, KLHL24, KLHL3, KLRB1, KRT72, KRT73, LIME1, LOC100128071,LOC100289511, LOC282997, LOC283070, LOC338799, LOC728392, LTBP3, MAL,MAP2K6, MDS2, MEGF6, MEGF6, MFGE8, MIR1909, MMP28, MOAP1, MXD4, MYADM,MYLIP, MYO15B, NFKBIZ, NLRC3, NLRP1, NOG, NR1D2, NR1D2, NR3C2, P2RY8,PBXIP1, PCSK5, PDE4D, PDZD4, PERI, PGAM2, PGCP, PHF1, PHF1, PIK3IP1,PIK3R5, PIM1, PION, PLCD1, PLCH2, PLCL1, PLEKHB1, PLK2, PLXDC1, PNRC1,PPP1R15A, ProSAPiPl, RAB37, RAP1GAP2, RARRES3, RASA3, RASGRP2, REM2,RGS1, RGS2, RNF125, SAMD3, SCML4, SEC31B, SIGIRR, SIK1, SLC2A3,SLC2A4RG, SLC2A4RG, SLC9A9, SLFN5, SMAD7, SMPD1, SNORA11, SORL1, SOX4,SULT1B1, SYNE1, SYTL1, TCEA3, TCF7, TCF7, TCP11L2, THEMIS, TMC8,TMEM63A, TMEM71, TMIGD2, TNFAIP3, TNNT3, TP53INP2, TPM2, TRANK1, TRIB2,TSC22D3, TSPAN18, TSPAN18, TSPAN32, TSSK3, TXK, TXNIP, UNC84B, UTRN,VIPR1, VSIG1, VSIG1, WHAMM, WNT10B, WNT7A, XAF1, XYLT1, XYLT1, YPEL2,YPEL3, YPEL5, ZBP1, ZBTB10, ZFP36, ZFP36L2, ZMAT1, ZNF331, and ZNF815.

Exemplary genes according to Table 2 upregulated in Treg v Teff at 16 hFC include ZBTB32, LRRC32, STAMBPL1, SNX10, LOC389333, ZNF193, GCNT1,FAS, GK3P, NTRK1, FREQ, IL1R1, CRADD, GNA15, RAB33A, IL18R1, CX3CR1,TNFRSF1B, APOBEC3G, FOXP3, SEPT11, CD70, IL1RL1, NIPA1, PANX2, CHST2,NEDD9, ACOT9, PDGFA, MAST4, TNFRSF8, PHLPP1, IL2RB, CTLA4, SYTL3,ZC3H12C, PTPRJ, UBASH3B, METRNL, PRDM1, SEPT3, TNFRSF18, WNT10A, CCR8,C18orf1, CSF1, CD80, GALNT4, GALNT4, IL1RL2, ADPRH, ZNF282, APOBEC3C,HS3ST3B1, EPAS1, RBKS, KAT2B, C9orf167, TYMP, IL1RAP, C2CD4A, CD68,ABHD4, MICAL2, C6orf145, DUSP16, LRIG1, CASK, EPSTI1, TNFRSF12A, IGSF3,SPATS2L, SPATS2L, MAF, CD58, KLHDC7B, ZBTB38, LAYN, IL1R2, HIP1, ITGB8,ITGB8, IKZF2, LGMN, XIRP1, GPR19, SAMD9L, PRF1, JAKMIP1, MGC29506,ADAMS, HLF, COL9A2, NDRG1, SAMHD1, AKAP5, RNF213, RNF213, APAF1, STX1A,SSH1, SSH1, CCRL2, CCR6, CSF2RB, HAVCR2, KLF5, MX1, ACTA2, OAS3, EMP1,CTNNAL1, MGC12916, CCL17, FOSL2, SAT1, TRPV2, PRIC285, SOCS2, ETV7,TIGIT, RASAL1, OPTN, MGST2, GPR68, MYO1G, PTPLA, TNFRSF11A, ANXA2, IRF5,C14orf139, CAPN2, LFNG, IL12RB1, MYO1E, GLRX, DENND3, ANXA2P2, NQO1,C10orf128, ANTXR2, ANTXR2, SLC26A11, FLVCR2, PREX1, SLC2A8, CDKN2A,TMEM149, SYT11, TOX, TOX2, FUT7, ANXA2P1, FAM129B, DFNB31, TMPRSS6,IL1RN, ISG15, CDKN1B, FAM129A, TST, HDAC9, TMEM110, SMPD1, CDKN1A,C17orf67, ANXA2P3, MPST, IRF7, LMCD1, SNX24, HMOX1, ATP2B4, FCER2, HPGD,RASGRP4, FAM164A, IFI6, FAM110C, XKRX, PBX4, NTNG2, CST7, BASP1,C14orf49, GLIPR1, DHRS2, TWIST1, SPSB1, CYTH4, CADM1, ITIH4, L00541471,CGA, LOC645166, PARP12, NINJ2, MICAL1, OAS1, HLA-DRB4, LGALS3, OASL,CORO2A, HLA-DRB3, KIAA1370, HERC6, STAC, MSC, CCR5, SUOX, RHOC,HLA-DQB2, PDE4A, LOC100302650, XAF1, FCRL3, RTKN2, GLIPR2, HLA-DRB1,IL13, P2RY10, IL10, CXCR6, LSP1, ACP5, SLC1A4, FXYD7, TRIB2, LMNA,HLA-DPA1, MEOX1, LGALS1, HLA-DRB5, IL10RA, HLA-DRA, CARD16, IL5, RGS1,HLA-DQA2, AKR1C3, IL4, HLA-DMA, GPR55, AQP3, MUSTN1, P2RY8, FANK1, IL9,CCNG2, ADAM12, LOC654342, IL17A, PPP2R2B, and FAM46C.

Exemplary genes according to Table 2 upregulated Treg vs Teff at 0 h FC4include C12orf75, SELPLG, SWAP70, RGS1, PRR11, SPATS2L, SPATS2L, TSHR,C14orf145, CASP8, SYT11, ACTN4, ANXA5, GLRX, HLA-DMB, PMCH, RAB11FIP1,IL32, FAM160B1, SHMT2, FRMD4B, CCR3, TNFRSF13B, NTNG2, CLDND1, BARD1,FCER1G, TYMS, ATP1B1, GJB6, FGL2, TK1, SLC2A8, CDKN2A, SKAP2, GPR55,CDCA7, S100A4, GDPD5, PMAIP1, ACOT9, CEP55, SGMS1, ADPRH, AKAP2, HDAC9,IKZF4, CARD17, VAV3, OBFC2A, ITGB1, CIITA, SETD7, HLA-DMA, CCR10,KIAA0101, SLC14A1, PTTG3P, DUSP10, FAM164A, PYHIN1, MYO1F, SLC1A4,MYBL2, PTTG1, RRM2, TP53INP1, CCR5, ST8SIA6, TOX, BFSP2, ITPRIPL1,NCAPH, HLA-DPB2, SYT4, NINJ2, FAM46C, CCR4, GBP5, C15orf53, LMCD1,MKI67, NUSAP1, PDE4A, E2F2, CD58, ARHGEF12, LOC100188949, FAS, HLA-DPB1,SELP, WEE1, HLA-DPA1, FCRL1, ICA1, CNTNAP1, OAS1, METTL7A, CCR6,HLA-DRB4, ANXA2P3, STAM, HLA-DQB2, LGALS1, ANXA2, PI16, DUSP4, LAYN,ANXA2P2, PTPLA, ANXA2P1, ZNF365, LAIR2, L00541471, RASGRP4, BCAS1, UTS2,MIAT, PRDM1, SEMA3G, FAM129A, HPGD, NCF4, LGALS3, CEACAM4, JAKMIP1,TIGIT, HLA-DRA, IKZF2, HLA-DRB1, FANK1, RTKN2, TRIB1, FCRL3, and FOXP3.

Exemplary genes according to Table 2 upregulated in Teff at 16 h v 0 hinclude AARS, ABCF2, ACOT7, ACTL6A, AHSA1, AIM2, AIMP2, ALAS1, ALDH1B1,ANKRD13B, APOL1, ARMCX3, ASPHD2, B3GNT5, B4GALT2, B4GALT5, BATF, BATF3,BCAT2, BCL2L1, BOP1, BTLA, BYSL, C11orf75, C15orf23, C15orf63, C16orf59,C17orf79, C17orf96, C1orf163, C3orf26, C4orf43, C8orf30A, C9orf64, CAD,CBR1, CCDC56, CCDC86, CCL20, CCL3, CCL3L1, CCL3L3, CCL4, CCL4L1, CCL4L2,CCNB1, CCND2, CCT3, CCT5, CCT6A, CCT7, CD109, CD200, CD274, CD3EAP,CD40LG, CD82, CDC20, CDC45L, CDC6, CDK4, CDT1, CENPM, CETN3, CHAC2,CHEK1, CISD1, CISH, CKS1B, COPB2, CORO1C, CSF2, CTNNA1, CTPS, CTTN,DARS2, DCAF12, DCTPP1, DHCR24, DKC1, DTL, E2F1, EBNA1BP2, ECE2, EDARADD,EEF1E1, EGR2, EIF2B3, EIF2S1, EIF5B, EIF6, ENO1, ESPL1, EXOSC3, EXOSC4,F5, F5, FABP5, FABP5L3, FADS1, FAM40B, FARSA, FASLG, FDPS, FKBP4, FKBP4,FOSL1, FREQ, GOS2, G3BP1, GALE, GAR1, GART, GEM, GEMIN6, GEMIN7, GFOD1,GINS1, GINS2, GLRX2, GNG8, GNPDA1, GPATCH4, GPN3, GPR171, GTF2H2D,HIVEP3, HMGCS1, HN1L, HNRNPAB, HSPD1, HSPE1, HYAL2, IARS, IER3, IFNG,IFRD2, IGFBP4, IL12RB2, IL15RA, IL17F, IL2, IL21, IL22, IL2RA, IL3,IRF4, IRF8, ISOC2, KCNK5, KEAP1, KIAA0020, KIAA0664, LAG3, LAPTM4B,LARP4, LIF, LOC286016, LOC344967, LOC442308, LOC728402, LRP8, LSM2, LTA,LYAR, MANF, MATK, MCM10, MCM2, MCM3, MCM4, METTL13, MIR1182, MIR155,MIR155HG, MIR621, MPV17L2, MPZL2, MRM1, MRPL12, MRPL15, MRPL17, MRPL35,MRPL51, MRPS17, MRPS23, MRTO4, MTCH2, MTHFD1L, MTHFD2, MYOF, NAB2,NDFIP2, NDUFAF1, NFE2L3, NFKBIL2, NLN, NME1, NME1-NME2, NOLC1, NOP16,NPTX1, NT5DC2, NUDCD1, NUP43, NUP62, OTUD7B, PACSIN3, PAICS, PAK1IP1,PAM, PDCD1, PDCD2L, PDIA4, PDIA6, PEA15, PFAS, PFDN6, PFDN6, PFKM, PFKP,PGAM1, PGAM4, PHB, PHF6, PKM2, PLAGL2, PNPO, POLD2, POLE2, POLR3K, POP1,PPIL1, PPP1R14B, PRDX1, PRDX3, PRDX4, PRMT1, PRMT5, PRSS23, PSAT1,PSMA3, PSMA5, PSMA6, PSMB3, PSMB5, PSMD1, PSMD11, PSMD14, PTGFRN, PTMS,PTRH1, PTRH2, PUS7, PYCR1, PYCRL, RARS, RBBP8, RCC1, RPF2, RPP25, RRP1,RRP9, RUVBL1, RUVBL2, SAMD4A, SCD, SDC4, SECTM1, SEH1L, SEMA7A, SFT2D1,SFXN1, SH2D2A, SHF, SHMT2, SIPA1L2, SLAMF1, SLC1A5, SLC27A2, SLC27A4,SLC29A1, SLC38A5, SLC39A14, SLC43A3, SLC6A9, SLCO4A1, SNORA18, SNORD17,SORD, SPR, SQLE, SRM, SRXN1, STIP1, STT3A, TALDO1, TAP1, TBKBP1, TBL3,TBX21, TIMM8B, TIMM8B, TIPIN, TMCC2, TMEM165, TMEM194A, TMEM208, TMEM97,TNF, TNFAIP8L2, TNFRSF4, TNFRSF9, TNFSF14, TOMM40, TPI1, TRIP10, TRIP13,TTLL12, TUBA1B, TUBB, TUBB, TUBB, TUBG1, TXN, TXNDC5, UBE2T, UCK2, UGDH,UHRF1, UMPS, UTP6, VDAC1, VDR, WARS, WDR12, WDR18, WDR3, WDR4, WDR77,YIF1A, YWHAG, ZBED2, ZDHHC16, ZNF593, ZNF607, and ZWINT.

Exemplary genes according to Table 2 upregulated in Treg 16h vs 0 h Fc8include AARS, ACOT7, AGRN, AHSA1, AIM2, AIMP2, ALAS1, ALDH1B1, APOL1,APOL2, B4GALT2, BATF, BATF3, BCL2A1, BCL2L1, BOP1, BYSL, C17orf96,C2CD4A, C5orf32, C9orf64, CCDC86, CCL17, CCL20, CCT5, CD3EAP, CD40LG,CD68, CD7, CDK2AP2, CDK4, CHAC2, CHPF, CISD1, CISH, COPB2, CRIM1, CSF1,CTLA4, CTSL1, CTTN, DCTPP1, DHCR24, EBI3, EBNA1BP2, ECE2, EDARADD, EGR2,EMP1, ENO1, EPAS1, EXOSC4, FABP5, FAH, FAM40B, FARSA, FKBP4, FKBP4,FLT1, FLT1, FOSL1, FREQ, G6PD, GALE, GART, GCLM, GEM, GK, GNPDA1, GPR56,HIVEP3, HMGCS1, HMOX1, HN1L, HSPA1A, HSPA1B, HSPD1, HSPE1, HYAL2, IER3,IFRD2, IKBIP, IL10, IL12RB2, IL13, IL15RA, IL17A, IL1R1, IL1R2, IL1RL2,IL1RN, IL2RA, IL3, IL4, IL4I1, IL5, IL9, IRF4, KCNK5, LAG3, LAPTM4B,LIF, LOC344967, LOC389333, LOC442308, LRRC32, LRRC61, LTA, LYAR, MANF,MATK, METRNL, METTL13, MGC29506, MICAL2, MIR1182, MIR155, MIR155HG,MLEC, MRPL12, MRTO4, MTHFD1L, MYOF, NAB2, NDFIP2, NDUFAF1, NKG7, NLN,NME1, NME1-NME2, NOP16, NPM3, NUDCD1, PAICS, PANX2, PDCD1, PDGFA, PDIA4,PDIA6, PFAS, PGAM4, PHB, PNPO, POP1, PPIL1, PPPDE2, PRDX1, PRDX3, PRDX4,PRKAR1B, PRMT1, PRMT5, PSAT1, PSMB5, PSMD1, PSMD11, PTGFRN, PTRH1, PUS7,PYCR1, RASAL1, RBBP8, RCC1, SC4MOL, SCD, SDC4, SECTM1, SEH1L, SEMA7A,SETP11, SERPINE2, SERPINH1, SH2D2A, SLC16A13, SLC16A3, SLC1A5, SLC27A2,SLC27A4, SLC29A1, SLC38A5, SLC39A1, SLC39A14, SLC43A3, SLCO4A1, SOCS1,SPHK1, SPINT1, SQLE, SRM, SRXN1, STIP1, STT3A, TBKBP1, TBX21, TMPRSS6,TNF, TNFRSF11A, TNFRSF12A, TNFRSF18, TNFRSF1B, TNFRSF4, TNFRSF8,TNFRSF9, TNFSF14, TOMM40, TRIP10, TTLL12, TUBB, TUBB, TUBB, TXN, TYMP,UCK2, UGDH, VDR, VTRNA1-3, WARS, WDR12, WDR4, WDR77, XIRP1, YWHAG,ZBED2, ZBTB32, ZDHHC16, and ZNF282.

An exemplary list of the T_(REG) genes upregulated at 16 h include AIM2,ALAS1, BATF, C5orf32, CCL17, CD40LG, CHAC2, CSF1, CTSL1, EBNA1BP2,EDARADD, EMP1, EPAS1, FABP5, FAM40B, FKBP4, FOSL1, GCLM, GK, GPR56,HMOX1, HSPD1, HSPE1, IKBIP, IL10, IL13, IL15RA, IL1RN, IL2RA, IL3, IL4,IL5, IL9, KCNK5, LTA, MANF, MIR1182, MIR155, MIR155HG, MYOF, NDUFAF1,NLN, NME1, NME1-NME2, PANX2, PDIA6, PGAM4, PPIL1, PPPDE2, PRDX4,PRKAR1B, PSMD1, PSMD11, PUS7, RBBP8, SLC27A2, SLC39A14, SLC43A3, SRXN1,STIP1, STT3A, TBX21, TNFRSF11A, TNFRSF1B, TNFRSF8, TNFRSF9, TXN, UCK2,VDR, VTRNA1-3, WDR12, YWHAG, ZDHHC16, and ZNF282. The upregulatedexpression may be determined, e.g., by measuring RNA levels for theindicated genes.

An exemplary list of the T_(EFF) genes upregulated at 16 h include AIM2,ALAS1, B4GALT5, BATF, C3orf26, C4orf43, CCL3, CCL4, CCT3, CCT7, CD40LG,CHAC2, CSF2, CTNNA1, EBNA1BP2, EDARADD, EEF1E1, EIF2B3, EIF2S1, FABP5,FAM40B, FKBP4, FOSL1, GFOD1, GLRX2, HSPD1, HSPE1, IFNG, IL15RA, IL21,IL2RA, IL3, KCNK5, KIAA0020, LARP4, LRP8, LTA, MANF, MIR1182, MIR155,MIR155HG, MTCH2, MYOF, NDUFAF1, NLN, NME1, NME1-NME2, OTUD7B, PAM,PDIA6, PEA15, PFKM, PGAM1, PGAM4, PPIL1, PRDX4, PRSS23, PSMD1, PSMD11,PSMD14, PTRH2, PUS7, RBBP8, RPF2, RPP25, SFXN1, SLC27A2, SLC39A14,SLC43A3, SORD, SPR, SRXN1, STIP1, STT3A, TBX21, TMCC2, TMEM165, TNFRSF9,TXN, TXNDC5, UCK2, VDR, WDR12, YWHAG, and ZDHHC16.

The Abbas gene sets compared the expression profiles of 17 immune celltypes and identified genes uniquely expressed in certain cell typesrelative to others. The select Abbas gene sets that were included in theGene Set Analysis are listed in Table 3 and include CD4+ T cells naïveand resting, CD8+ T cells naïve and resting, helper Th1 at 12 hours,helper Th1 at 48 hours, helper Th2 at 12 hours, helper Th2 at 48 hours,memory T resting (naïve) cells, and memory T activated cells.

TABLE 3 Table 3: Gene sets comprised of gene up or down regulatedresting and activated T-cell subtypes (Abbas data set) Gene Set Numberof genes Downregulated CD8 vs CD4 Naïve T-cells 200 Downregulated NaïveCD4 vs 12 H activated Th1 200 Downregulated Naïve CD4 vs 48 H activatedTh1 200 Downregulated Naïve CD4 vs 12 H activated Th2 200 DownregulatedNaïve CD4 vs 48 H activated Th2 200 Downregulated Th1 vs Th2 12 Hactivated 200 Downregulated Th1 vs Th2 48 H activated 200 Downregulatedunstimulated vs stimulated memory 200 T-cells Upregulated CD8 vs CD4Nave T-cells 200 Upregulated Naïve CD4 vs 12 H activated Th1 200Upregulated Naïve CD4 vs 48 H activated Th1 200 Upregulated Naïve CD4 vs12 H activated Th2 200 Upregulated Naïve CD4 vs 48 H activated Th2 200Upregulated Th1 vs Th2 12 H activated 200 Upregulated Th1 vs Th2 48 Hactivated 200 Upregulated unstimulated vs stimulated memory 200 T-cells

The Gattinoni gene sets compared the expression profiles of various CD8+memory T cell subsets. Specifically, immune cells were isolated fromhealthy donors and the following CD8+T memory subsets were purified:T_(N) (naïve), T_(SCM) (memory stem cells), T_(CM) (central memory),T_(EM) (effector memory). Gene sets were defined by comparing betweenall pairs of groups (e.g. T_(SCM) vs. T_(N)) and by identifying thosegenes that either progressively increased or decreased across the 4conditions in order from T_(N)→T_(SCM)→T_(CM)→T_(EM). The select genesets from Gattinoni et al. that were considered in the Gene Set Analysisare tabulated in Table 4.

TABLE 4 Table 4: Gene sets comprised of gene up or down regulatedresting and activated T-cell subtypes (Gattinoni data set) Gene SetNumber of genes T_(CM) vs T_(EM) 29 T_(N) vs T_(CM) 148 T_(N) vs T_(EM)212 T_(SCM) vs T_(CM) 19 T_(SCM) vs T_(EM) 75 T_(SCM) vs T_(N) 73Progressively down 208 Progressively up 32

Each gene set (e.g., ALL and CLL RNAseq gene sets, Szabo gene sets,Abbas gene sets, and Gattinoni gene sets) was evaluated to determine itsassociation with subject response (i.e., CR, PR, or NR) in the followingmanner: a meta-gene was calculated for each subject, where the meta-genescore for subject j was defined as

m _(j)=Σ_(i=G) ¹ x _(ij)−μ(x _(.j))/σ(x _(.j))

where x_(ij) is the expression value of gene i in subject j for a givengene set n=1, . . . , G; μ(x_(.j)) is the mean of genes 1, . . . , G insubject j; and σ(x_(.j)) is the standard deviation of genes 1, . . . , Gin subject j.

A 3-group statistical model was applied to each gene set to determinewhether the meta-gene was statistically different between the CLLproduct CRs, PRs, and NRs. A schematic illustrating this approach isgiven in FIG. 2B. CRs are more like resting T_(EFF) cells, whereas NRare more like activated T_(EFF) cells. CTL019 NR samples are in a moreactivated state than CR samples. Gene sets that were found to besignificantly altered and predictive of patient response to CD19CAR-expressing cell (e.g., T cell, NK cell) therapy (e.g., CTL019) arelisted in Table 5.

TABLE 5 Table 5: Gene sets that are enriched in CLL samples Prod- Aphe-Gene Set Source CRs NRs uct resis T_(EFF) 16 h vs 0 h Szabo T_(EFF) ohT_(EFF) 16 h X X T_(REG) 16 h vs 0 h Szabo T_(REG) oh T_(REG) 16 h X XT_(REG) vs T_(EFF) 16 h Szabo T_(EFF) T_(REG) X X Naïve CD4 vs AbbasNaïve Th1 X X 12 H act Th1 CD4 Naïve CD4 vs Abbas Naïve Th2 X X 12 H actTh2 CD4 Naïve CD4 vs Abbas Naïve Th1 X X 48 H act Th1 CD4 Naïve CD4 vsAbbas Naïve Th2 X X 48 H act Th2 CD4 Unstim vs stim Abbas Unstim-Stimulated X memory ulated T_(SCM) vs T_(EM) Gattinoni T_(SCM) T_(EM) XT_(SCM) vs T_(CM) Gattinoni T_(SCM) T_(CM) X T_(CM) vs T_(EM) GattinoniT_(EM) T_(CM) X Progressively Gattinoni Early Late stage X X down stage

FIG. 3 depicts an exemplary schematic of memory T cell precursors andsubsets. Without wishing to be bound by a particular theory, the stateof memory T cells in CTL019 samples is likely a major component ofresponse.

For a subset of the patients in the manufactured product study describedin Example 1, whole genome RNAseq was also performed on T cellscollected by apheresis. The gene sets described above were evaluated inthese 14 apheresed samples (2 CRs, 3 PRs, and 9 NRs). Gene sets thatwere found to be significantly altered and predictive of patientresponse to CTL019 therapy are tabulated in Table 5.

Whole genome RNAseq was performed on 7 ALL manufactured product CRsamples and 4 ALL apheresis CR samples. Meta-gene scores for each geneset were calculated for the ALL samples as described above for the CLLsamples. Gene sets with meta-gene scores correlating with the expectedpattern of response in product and apheresis samples (ALL→CLL CR→CLLPR→CLL NR) are tabulated in Table 6. Gene sets marked with * in Table 6are also correlated with response in apheresis samples.

TABLE 6 Table 6: Gene sets correlating to response over product ALL andCLL samples (ALL → CLL CR → CLL PR → CLL NR) Gene Set Downregulated Tregvs Teff 0 h (FC3 p < 0.05)* Downregulated Treg vs Teff 16 h (FC3 p <0.05) Upregulated Treg vs Teff 0 h (FC4 p < 0.05)* Upregulated Treg vsTeff 16 h (FC4 p < 0.05) TCMvsTEM down gene set TCMvsTEM up gene setTNvsTCM down gene set* TNvsTCM up gene set* TNvsTEM down gene set*TNvsTEM up gene set* TSCMvsTCM down gene set* TSCMvsTCM up gene set*TSCMvsTEM up gene set* TSCMvsTN down gene set* TSCMvsTN up gene set*Progressively down* Progressively up* Downregulated CD8 vs CD4 NaveT-cells* Upregulated Naïve CD4 vs 12 H activated Th1 Upregulated NaïveCD4 vs 48 H activated Th1 Upregulated Naïve CD4 vs 12 H activated Th2Upregulated Naïve CD4 vs 48 H activated Th2* Downregulated Th1 vs Th2 12H activated

For example, the meta-gene score for the gene set comprised of genesupregulated in T_(SCM) in comparison to T_(CM) is found to be correlatedwith response in both apheresis and product samples, FIG. 4. Themeta-gene scores from healthy donor samples with manufactured productare included in the plot to serve as a reference point. The x-axis issamples by response group where a=apheresis and p=product. The y-axis isnormalized meta-gene expression scores. Gene sets enriched in CLL CRs(e.g., CTL019 CRs) are also enriched in acute lymphoblastic leukemias(ALLs). ALL and CLL CRs are enriched in T stem cell (T_(SCM)) subsetspecific genes, whereas CLL PRs and NRs are enriched in T central memory(T_(CM)) subset genes. The same pattern is seen in apheresis as inproduct samples. ALL expression patters are most similar to CLL CRs andare even more extreme in the direction of resting/unstimulated/earlymemory T cells.

FIG. 5A depicts an exemplary result from a Principle Component Analysis(PCA) of CTL019 samples. This exemplary PCA result illustrates that CRs,ALL and Normal samples cluster separately from PRs and NRs. FIG. 5Bdepicts an exemplary result from a PCA of CTL019 and apheresis samples.This exemplary PCA result illustrates that CRs, ALL and Normal samplescluster separately from PRs and NRs and from the apheresis cluster.

FIG. 6 depicts an exemplary schematic depicting immunophenotyping ofapheresis and product samples.

Manufactured CTL019 product (e.g., genetically engineeredCAR19-expressing T cells obtained from CLL patients) classified ascomplete responders (CR), partial responders (PR), non-responders (NR),or pending were assessed for expression of immune checkpoint inhibitormolecules, such as PD-1, LAG3, and TIM3.

CD 19 CAR-expressing cells (e.g., T cells, NK cells) from CLL patients(e.g., manufactured product) with different responses to CAR-expressingcell therapy were analyzed by flow cytometery to determine percentagesof CD4+ and CD8+ T cells. The CD19 CAR-expressing cells were from:patients that responded to CAR-expressing cell therapy (CR) (n=5);patients that partially responded to CAR-expressing cell therapy (n=8),patients that did not respond to CAR-expressing cell therapy (NR)(n=19); and patients that were pending, e.g., not yet assigned to agroup (NA) (n=3). Cells were labeled with antibodies that specifiallyrecognize CD4, CD8, the CAR19 molecule, and immune checkpoint moleculesPD-1, LAG3, and TIM3, and secondary antibodies conjugated tofluoresceins, according to standard methods for flow cytometry analysisknown in the art. Expression of each marker, e.g., CD4+, CD8+, etc., wasdeterined by flow cytometry analysis software, and subpopulations (e.g.,CD4+ T cells, CD8+T cells, or CAR19-expressing T cells) were furtheranalyzed fro the expression of immune checkpoint molecules PD-1, LAG3,and TIM3.

Using the methods and analysis described above, the percentage ofCD4-expressing cells and CD8-expressing cells was determined for eachpatient in each response group. As described above, 36 manufacturedCTL019 samples from CLL patients were analyzed, and included 5 CR, 8 PR,19NR and 3 pending. FIG. 7A depicts an exemplary result illustratingpercent CD4+ cells and patient response. Partial responders were shownto have a statistically significant greater percentage of CD4+ cells.FIG. 7B depicts an exemplary result illustrating percent CD8+ cells andpatient response. Complete responders were shown to have a statisticallysignificant great percentage of CD8+ cells.

An example of the flow cytometry profiles analysis used to determinesurface marker expression is shown in FIGS. 8A and 8B. Cells expressingCD4+ were determined using flow cytometry, and were further analyzed forCAR19 and PD-1 expression, such that the x-axis of the profiles indicateCAR19 expression (the top left (Q5) and bottom left (Q8) quadrants showthe CAR19-negative CD4+ cells, while the top right (Q6) and bottom right(Q7) quadrants show the CAR19-expressing CD4+ cells) and the y-axisshows PD-1 expression (the bottom left (Q8) and right (Q7) quadrantsshow the PD-1 negative CD4+ cells and the top left (Q5) and right (Q6)quadrants show the PD-1− expressing CD4+ cells). In the CD4+ populationfrom a CAR-expressing cell (e.g., T cell, NK cell) responder, 44.7% ofthe CD4+ cells overall expressed PD-1, and about 22.3% of theCAR19-expressing cells were PD-1 positive, while 27.2% ofCAR19-expressing cells were PD-1 negative (FIG. 8A). In contrast, in theCD4+ population from a non-responder, there was a significant decreasein CAR19-expressing cells overall (about 15.3% compared to the 49.5% inCR), with 14.7% of the CAR19-expressing cells being PD-1 positive whileonly 0.64% were PD-1 negative (FIG. 8B). Comparison between the profilesin FIG. 8A and FIG. 8B shows that a much higher percentage of the CD4+cells from a non-responder express PD-1 (about 92.9%) compared to theCAR-expressing cell responder (about 44.7%).

Using the methods and analysis described above, the percentage of PD-1expressing (PD-1+) cells of the CD4+ population and the CD8+ populationwas determined for each patient in each response group. Non-responderswere shown to have a greater percentage of PD-1+ cells in both theCD4+(FIG. 8C) and CD8+(FIG. 8D) populations compared to those thatresponded to CAR therapy (CR); the increase of average PD-1 percentagewas statistically significant for both CD4+ and CD8+ populations.Partial responders (PR) exhibited higher percentages of PD-1+ cells thanresponders (CR) in both CD4+(FIG. 8C) and CD8+(FIG. 8D) populations.

Further analysis was performed to determine the distribution of cellsexpressing PD-1, LAG3, and TIM3 from patients with different responsesto CAR therapy. Representative cell profile analysis for PD-1, LAG3, andTIM3 expression in the CD4+ population are shown in FIG. 9 and FIG. 10.The cell populations were first analyzed for CAR19+ expression. TheCAR19+ population was then analyzed for PD-1 and LAG3 expression (FIG.9) or PD-1 and TIM-3 expression (FIG. 10). In the LAG3+ population froma CAR-expressing cell (e.g., T cell, NK cell) responder, 36.1% of theCAR19+ cells overall expressed PD-1, and about 7.3% of theLAG3-expressing cells were PD-1 positive, while 5.9% of LAG3-expressingcells were PD-1 negative (FIG. 9). In contrast, in the CAR19+ populationfrom a non-responder, there was a significant increase inLAG3-expressing cells overall (about 69.7% compared to the 13.2% in CR),with 67.3% of the LAG3-expressing cells being CAR19+ positive while only2.41% were PD-1 negative (FIG. 9). Comparison between the CR and NR flowcytometry profiles in FIG. 9 show that a much higher percentage of theLAG3+ cells from a non-responder express PD-1 (about 67.3%) compared tothe CAR-expressing cell (e.g., T cell, NK cell) responder (about 7.3%).

Using the methods and analysis described above, the percentage of PD-1and LAG-3 expressing (PD-1+/LAG-3+) cells of the CAR19+ population wasdetermined for each patient in each response group. Non-responders wereshown to have a greater percentage of PD-1+/LAG-3+ cells in the CAR19+populations compared to those that responded to CAR therapy (CR) (FIG.9); the increase of average PD-1/LAG-3 percentage was statisticallysignificant for the CAR19+ population. Partial responders (PR) exhibitedhigher percentages of PD-1+/LAG-3+ cells than responders (CR) in theCAR19+(FIG. 9) population. In an embodiment, NR products exhibit anexhausted phenotype of PD1+CAR+ and co-expression of LAG3.

Next, the CAR19+ population was analyzed for PD-1 and TIM-3 expression(FIG. 10). In the TIM+ population from a CAR-expressing cell (e.g., Tcell, NK cell) responder, 28.5% of the CAR19+ cells overall expressedPD-1 (FIG. 10). In contrast, in the CAR19+ population from anon-responder, there was a significant increase in TIM3+/PD1+ cells,with 83.3% of the CAR19+-expressing cells being TIM3+/PD1+(FIG. 10).

Using the methods and analysis described above, the percentage of PD-1and TIM-3 expressing (PD-1+/TIM-3+) cells of the CAR19+ population wasdetermined for each patient in each response group. Non-responders wereshown to have a greater percentage of PD-1+/TIM-3+ cells in the CAR19+populations compared to those that responded to CAR therapy (CR) (FIG.10); the increase of average PD-1/TIM-3 percentage was statisticallysignificant for the CAR19+ population. Partial responders (PR) exhibitedhigher percentages of PD-1+/TIM-3+ cells than responders (CR) in theCAR19+(FIG. 10) population. In an embodiment, NR products exhibit anexhausted phenotype of PD1+CAR+ and co-expression of TIM3.

Cells expressing CD4+ and CD8+ were determined using flow cytometry, andwere further analyzed for CD27+ expression. Using the methods andanalysis described above, the percentage of CD27 expressing (CD27+)cells of the CD4+ population and the CD8+ population was determined foreach patient in each response group. Complete responders (CR) andpartial responders (PR) were shown to have a greater percentage of CD27+cells in both the CD4+(FIG. 11A) and CD8+(FIG. 11B) populations comparedto non-responders (NR); the increase of average CD27 percentage wasstatistically significant for both CD4+ and CD8+ populations. Partialresponders (PR) exhibited higher percentages of CD27+ cells thancomplete responders (CR) in CD4+(FIG. 11A) populations. Completeresponders (CR) exhibited higher percentages of CD27+ cells than partialresponders (PR) in CD8+(FIG. 11B) populations. In an embodiment, CD27levels in a CAR product correlate with patient response. In anembodiment, CRs CD8+ cells display higher percentages of CD27+ cells ascompared to PRs and NRs.

FIG. 12 depicts an exemplary multi-color flow cytometry analysis resultidentifying correlates of response in apheresis samples. 26 apheresedsamples from CLL patients were analyzed. Samples included 4 CR, 6 PR,14NR and 1 patient was not infused.

FIG. 13 depicts an exemplary multi-color flow cytometry analysis resultillustrating a correlation between a younger T cell phenotype andresponse to CTL019 therapy. These data demonstrate that the percentageof CD27+CD45RO− in CD8+ T cells is predictive of which CLL patients willundergo a complete response to CTL019.

FIG. 14 depicts an exemplary analysis of apheresis in a human patientprior to CTL019 therapy. Exemplary results illustrate that while patient1000-00045 presented with very few T cells, 27% of the T cells wereCD8+CD27+CD45RO−.

FIG. 15 depicts an exemplary result of a patient response (patient1000-00045) to CTL019 therapy. CD8+CD27+CD45RO− T cells were a positivepredictor of the patient response to CTL019 therapy. These exemplaryresults illustrate that a good prognostic phenotype in apheresis is ahigh percentage of CD8+CD27+CD45RO− T cells (young phenotype). A poorprognostic phenotype in CTL019 product is a high percentage of PD1+CAR+and LAG3+ or TIM3+ T cells (exhausted phenotype).

Significant gene sets from the analyses above were refined to a subsetof genes within the gene sets that are significantly differentiallyexpressed between ALL CRs/CLL CRs and CLL NRs as well as following theexpected expression pattern of increasing or decreasing from ALL→CLLCR→CLL PR→CLL NR. An exemplary listing of genes that were significantlydifferentially expressed are listed in Table 7A. Table 7A is anexemplary list of biomarkers whose expression values predict patientresponse to CTL019 therapy. Table 7A was further refined to produce aflow cytometry biomarker gene panel by selecting for genes that are cellsurface markers. Exemplary cell surface genes that predict patientresponse to CTL019 therapy are shown in Table 8.

TABLE 7A Table 7A: Exemplary genes that predict patient response toCTL019 therapy Gene Unigene Accession No. ABCA7 Hs.134514 NM_019112ABTB1 Hs.107812 NM_172027, NM_172028, NM_032548 ACOT9 Hs.298885NM_001037171, NM_001033583 ACTA2 Hs.500483 NM_001141945, NM_001613ADAMTS10 Hs.657508 NM_030957 ADD3 Hs.501012 NM_016824, NM_019903,NM_001121 ADPRH Hs.99884 NM_001125 AEBP1 Hs.439463 NM_001129 AESHs.515053 NM_198970, NM_198969, NM_001130 AIM2 Hs.281898 NM_004833 ALAS1Hs.476308 NM_199166, NM_000688 ALPK1 Hs.652825 NM_025144, NM_001102406ALS2CL Hs.517937 NM_147129, NM_182775 AMD1 Hs.159118 NM_001634,NM_001033059 ANKRD55 Hs.436214 NM_024669, NM_001039935 ANKZF1 Hs.437647NM_018089, NM_001042410 ANTXR2 Hs.162963, NM_001145794, NM_058172Hs.720941 ANXA2 Hs.591361, NM_004039, NM_001136015, Hs.546235,NM_001002858, NM_001002857 Hs.511605 ANXA2P2 Hs.534301 AP1G2 Hs.343244NM_003917 AP1M1 Hs.71040 NM_032493, NM_001130524 AP2A2 Hs.19121NM_012305 APAF1 Hs.552567 NM_181869, NM_181868, NM_013229, NM_001160,NM_181861 APBA2 Hs.721380, NM_001130414, NM_005503 Hs.618112 APBB3Hs.529449 NM_133174, NM_133173, NM_133172, NM_006051 AQP3 Hs.234642NM_004925 ARFGAP2 Hs.436204 NM_032389 ARHGAP33 Hs.515364 NM_052948ARHGEF1 Hs.631550 NM_004706, NM_199002, NM_198977 ARHGEF11 Hs.516954NM_198236, NM_014784 ARHGEF18 Hs.465761 NM_001130955, NM_015318 ARL4CHs.723194, NM_005737 Hs.111554 ARPC5L Hs.132499 NM_030978 ARRB1Hs.625320, NM_020251, NM_004041 Hs.503284 ARRDC2 Hs.515249 NM_015683,NM_001025604 ARSB Hs.604199, NM_000046, NM_198709 Hs.149103 ATOX1Hs.125213 NM_004045 ATP13A3 Hs.529609 NM_024524 ATP1B3 Hs.477789NM_001679 ATP2A2 Hs.506759 NM_001135765, NM_170665, NM_001681 ATP2B4Hs.343522, NM_001001396, NM_001684 Hs.511311 ATP8B4 Hs.511311 NM_024837ATXN7L3B Hs.744849 NM_001136262 AVEN Hs.555966 NM_020371 B4GALT5Hs.370487 NM_004776 BATF Hs.509964 NM_006399 BCL9L Hs.414740 NM_182557BENDS Hs.475348 NM_024603 BEX4 Hs.184736 NM_001080425, NM_001127688 BIN1Hs.193163 NM_139350, NM_139348, NM_139349, NM_139343, NM_004305,NM_139345, NM_139344, NM_139347, NM_139351, NM_139346 BNIP3L Hs.131226NM_004331 BTN3A1 Hs.191510 NM_001145008, NM_007048, NM_194441,NM_001145009 C10orf128 Hs.385493 NM_001010863 C11orf10 Hs.437779NM_014206 C11orf21 Hs.559181 NM_001142946 C11orf35 Hs.669395 NM_173573C12orf5 Hs.504545 NM_020375 C16orf54 Hs.331095 NM_175900 C16orf74Hs.461655 NM_206967 C17orf48 Hs.47668 NM_020233 C17orf67 Hs.658949NM_001085430 C19orf29 Hs.267446 NM_001080543, NM_021231 C1QBP Hs.555866NM_001212 C20orf11 Hs.353013 NM_017896 C20orf112 Hs.516978 NM_080616C21orf2 Hs.517331 NM_004928 C2orf67 Hs.591638, NM_152519 Hs.282260C3orf26 NM_001167924, NM_032359 C4orf43 NM_018352 C5orf13 Hs.483067,NM_001142475, NM_001142476, Hs.36053, NM_004772, NM_001142482, Hs.694860NM_001142477, NM_001142483, NM_001142478, NM_001142474, NM_001142481,NM_001142479, NM_001142480 C5orf30 Hs.482976 NM_033211 C5orf32 Hs.529798NM_032412 C5orf39 Hs.529385, NM_001014279 Hs.721020 CABIN1 Hs.517478NM_012295 CACHD1 Hs.443891 NM_020925 CADM1 Hs.370510 NM_014333,NM_001098517 CAPG Hs.516155 NM_001747 CAPS Hs.584744 NM_004058,NM_080590 CASK Hs.495984 NM_001126054, NM_001126055, NM_003688 CBX4Hs.405046 NM_003655 CCDC47 Hs.202011 NM_020198 CCL17 Hs.546294 NM_002987CCL3 Hs.514107 NM_002983 CCL4 Hs.75703 NM_002984 CCR1 Hs.301921NM_001295 CCT2 Hs.189772 NM_006431 CCT3 Hs.491494 NM_001008800,NM_005998, NM_001008883 CCT7 Hs.368149 NM_001009570, NM_006429,NM_001166284, NM_001166285 CD248 Hs.195727 NM_020404 CD40LG Hs.592244NM_000074 CD58 Hs.34341 NM_001144822, NM_001779 CD70 Hs.715224,NM_001252 Hs.501497 CD80 Hs.838 NM_005191 CDC123 Hs.412842 NM_006023CDC25B Hs.153752 NM_004358, NM_021872, NM_021873 CDC42BPG Hs.293590NM_017525 CDK7 Hs.184298 NM_001799 CDKN1A Hs.370771 NM_078467, NM_000389CDKN2A Hs.512599 NM_058197, NM_058195, NM_000077 CERK Hs.200668NM_022766 CFP Hs.53155 NM_001145252, NM_002621 CHAC2 Hs.585944NM_001008708 CHI3L2 Hs.514840 NM_001025199, NM_001025197, NM_004000CHMP7 Hs.5019 NM_152272 CLDND1 Hs.531371 NM_001040181, NM_001040183,NM_001040200, NM_001040199, NM_001040182, NM_019895 CLTC Hs.491351NM_004859 CNN3 Hs.483454 NM_001839 CNOT8 Hs.26703 NM_004779 CNPY3Hs.414099 NM_006586 COQ3 Hs.713623 NM_017421 CSF1 Hs.591402 NM_000757,NM_172212, NM_172211, NM_172210 CSF2 Hs.1349 NM_000758 CSNK2A1Hs.654675, NM_001895, NM_177560, NM_177559 Hs.644056 CST7 Hs.143212NM_003650 CTC1 Hs.156055 NM_025099 CTDSP1 Hs.444468 NM_182642, NM_021198CTDSP2 Hs.524530 XM_001720210, XM_001722552, XM_002344384, XM_001725997,NM_005730 CTNNA1 Hs.656653, NM_001903 Hs.445981 CTSL1 Hs.418123NM_001912, NM_145918 CUL9 Hs.485434 NM_015089 CUTA Hs.520070NM_001014433, NM_001014840, NM_015921, NM_001014838, NM_001014837 CYFIP1Hs.26704 NM_014608, NM_001033028 CYP2J2 Hs.152096 NM_000775 DBPHs.414480, NM_001352 Hs.528006 DCAF11 Hs.525251 NM_001163484, NM_181357,NM_025230 DCBLD2 Hs.203691 NM_080927 DCHS1 Hs.199850 NM_003737 DCTN6Hs.158427 NM_006571 DDX10 Hs.591931 NM_004398 DENND2D Hs.557850NM_024901 DENND5A Hs.501857 NM_015213 DERL1 Hs.241576 NM_001134671,NM_024295 DFNB31 Hs.93836 NM_001083885, NM_015404 DGKD Hs.471675NM_152879, NM_003648 DGKZ Hs.502461 NM_001105540, NM_003646, NM_201533,NM_201532 DHRS2 Hs.272499 NM_182908, NM_005794 DIABLO Hs.169611NM_138929, NM_019887 DNAJB6 Hs.490745 NM_005494, NM_058246 DPEP2Hs.372633 NM_022355 DUSP22 Hs.29106 NM_020185, XM_001718070 E2F6Hs.603093 NM_198256 EBNA1BP2 Hs.346868 NM_006824, NM_001159936 EDARADDHs.352224 NM_080738, NM_145861 EED Hs.503510 NM_152991, NM_003797 EEF1E1Hs.602353, NM_004280, NM_001135650 Hs.723203 EGFL6 Hs.12844NM_001167890, NM_015507 EHD1 Hs.523774 NM_006795 EIF2B3 Hs.533549NM_001166588, NM_020365 EIF2S1 Hs.151777 NM_004094 ELL2 Hs.708710,NM_012081 Hs.192221 EMP1 Hs.436298 NM_001423 EPAS1 Hs.468410 NM_001430EPHA4 Hs.371218 NM_004438 EPHX1 Hs.89649 NM_001136018, NM_000120 EPPK1Hs.200412 NM_031308 ERGIC2 Hs.339453 NM_016570 ERGIC3 Hs.472558NM_015966, NM_198398 ERP29 Hs.75841 NM_001034025, NM_006817 ETFAHs.39925 NM_001127716, NM_000126 ETNK1 Hs.29464 NM_001039481, NM_018638ETV7 Hs.272398 NM_016135 FAAH Hs.720143 NM_001441 FABP5 Hs.408061NM_001444 FAF2 Hs.484242 NM_014613 FAIM3 Hs.723317, NM_001142472,NM_001142473, Hs.58831 NM_005449 FAM117B Hs.471130 NM_173511 FAM134BHs.711125 NM_001034850, NM_019000 FAM13A Hs.97270 NM_014883,NM_001015045 FAM193B Hs.484289 NM_019057 FAM40B Hs.489988 NM_020704,NM_001134336 FAM63A Hs.723127 NM_018379, NM_001163260, NM_001163259,NM_001163258, NM_001040217 FAM65B Hs.559459 NM_014722, NM_015864 FANCLHs.720331 NM_001114636, NM_018062 FANK1 Hs.352591 NM_145235 FAR2Hs.298851 NM_018099 FAU Hs.387208 NM_001997 FCER1G Hs.433300 NM_004106FCER2 Hs.465778 NM_002002 FCGBP Hs.111732 NM_003890, XM_001717543 FCHO1Hs.96485 NM_001161358, NM_001161357, NM_001161359, NM_015122 FCRL3Hs.292449 NM_052939 FGD3 Hs.411081 NM_033086, NM_001083536 FGF9 Hs.111NM_002010 FKBP4 Hs.713721, NM_002014 Hs.524183 FLOT2 Hs.514038 NM_004475FLT3LG Hs.428 NM_001459 FLVCR2 Hs.615289, NM_017791 Hs.509966 FOSL1Hs.283565 NM_005438 FOSL2 Hs.596972, NM_005253 Hs.220971 FRAT1 Hs.126057NM_005479 GAL3ST4 Hs.44856 NM_024637 GALNT4 Hs.713979, NM_003774Hs.25130 GCLM Hs.315562 NM_002061 GCNT1 Hs.521568 NM_001490,NM_001097633, NM_001097635, NM_001097634, NM_001097636 GFOD1 Hs.484686NM_018988 GFPT1 Hs.580300 NM_002056 GIPC3 Hs.266873 NM_133261 GKHs.1466, NM_001128127, NM_000167, Hs.654557 NM_203391 GLRX2 Hs.458283NM_016066, NM_197962 GMEB2 Hs.473286 NM_012384 GNAI1 Hs.134587 NM_002069GPA33 Hs.651244 NM_005814 GPD1L Hs.82432 NM_015141 GPKOW Hs.503666NM_015698 GPR125 Hs.99195 NM_145290 GPR56 Hs.513633 NM_001145773,NM_001145774, NM_001145771, NM_001145772, NM_005682, NM_201525,NM_001145770, NM_201524 GPSM3 Hs.520046 NM_022107 GRAP Hs.567416NM_006613 GRASP Hs.407202 NM_181711 GTF2A2 Hs.512934 NM_004492 HAVCR1Hs.129711 NM_001099414, NM_012206 HBS1L Hs.378532 NM_001145207,NM_001145158, NM_006620 HDAC9 Hs.196054 NM_014707, NM_178423, NM_178425,NM_058176, NM_058177 HIGD1A Hs.711098, NM_014056, NM_001099669,Hs.593134, NM_001099668 Hs.7917 HIP1 Hs.329266, NM_005338 Hs.619089HLA-DMA Hs.351279 NM_006120 HLA-DPA1 Hs.347270 NM_033554 HLA-DQA2Hs.591798 NM_020056 HLA-DQB2 Hs.719990 NM_001198858, NM_001300790HLA-DRA Hs.520048 NM_019111 HLA-DRB1 Hs.716081, NM_002124, NM_021983,Hs.696211, XM_002346251 Hs.723344, Hs.534322 HLA-DRB5 Hs.534322NM_002125 HLF Hs.196952 NM_002126 HMOX1 Hs.517581 NM_002133 HSPD1Hs.595053, NM_199440, NM_002156 Hs.723164 HSPE1 Hs.1197 NM_002157 HYIHs.709864 NM_031207 ICAM3 Hs.654563 NM_002162 IDUA Hs.89560 NM_000203IER2 Hs.501629 NM_004907 IFNAR2 Hs.708195 NM_207584, NM_207585,NM_000874 IFNG Hs.856 NM_207585 IGF1R Hs.643120, NM_000875 Hs.714012IGSF3 Hs.171057 NM_001007237, NM_001542 IGSF9B Hs.204121 NM_014987 IKBIPHs.252543 NM_201612, NM_201613, NM_153687 IL10 Hs.193717 NM_000572IL11RA Hs.591088 NM_004512, NM_147162, NM_001142784 IL13 Hs.845NM_002188 IL15RA Hs.524117 NM_002189, NM_172200 IL1RAP Hs.478673NM_134470, NM_001167930, NM_001167928, NM_001167929, NM_002182 IL1RL1Hs.66 NM_003856, NM_016232 IL1RN Hs.81134 NM_000577, NM_173841,NM_173842, NM_173843 IL21 Hs.567559 NM_021803 IL2RA Hs.231367 NM_000417IL2RB Hs.474787 NM_000878 IL3 Hs.694 NM_000588 IL4 Hs.73917 NM_000589,NM_172348 IL5 Hs.2247 NM_000879 IL6ST Hs.532082 NM_002184, NM_175767 IL9Hs.960 NM_000590 ING4 Hs.524210 NM_001127583, NM_001127582,NM_001127586, NM_001127585, NM_001127584, NM_016162 INPP5A Hs.523360,NM_005539 Hs.715308 INTS1 Hs.532188 NM_001080453 IRF2BP2 Hs.350268NM_001077397, NM_182972 ISOC1 Hs.483296 NM_016048 ITGA6 Hs.133397NM_001079818, NM_000210 ITPKB Hs.528087, NM_002221 Hs.659396 ITPR3Hs.65758 NM_002224 JAKMIP1 Hs.479066 NM_144720, NM_001099433 KAT8Hs.533803 NM_032188, NM_182958 KCNK5 Hs.444448 NM_003740 KCTD12Hs.644125 NM_138444 KIAA0020 Hs.493309 NM_014878 KIAA0141 Hs.210532NM_014773, NM_001142603 KIAA0664L3 Hs.715792 KIAA0748 Hs.33187NM_001098815, NM_001136030 KIAA1257 Hs.518247 NM_020741 KIAA1279Hs.279580 NM_015634 KIAA1683 Hs.313471 NM_025249, NM_001145305,NM_001145304 KIAA1797 Hs.136247 NM_017794 KIF3A Hs.43670 NM_007054 KITHs.479754 NM_001093772, NM_000222, XM_001724747, XM_936229 KLF2Hs.107740 NM_016270 KLF3 Hs.298658 NM_016531 KPNA3 Hs.527919 NM_002267KRT72 Hs.662013 NM_080747, NM_001146226, NM_001146225 KRT73 Hs.55410NM_175068 LAIR1 Hs.572535 NM_002287, NM_021706 LARP4 Hs.26613 NM_199188,NM_199190, NM_052879, NM_001170808, NM_001170803, NM_001170804 LDLRAP1Hs.590911 NM_015627 LEF1 Hs.555947 NM_001166119, NM_001130713,NM_001130714, NM_016269 LGMN Hs.18069 NM_001008530, NM_005606 LIMA1Hs.525419 NM_001113547, NM_001113546, NM_0016357, NM_017806 LIME1Hs.233220 NM_017806 LMBR1L Hs.272838 NM_018113 LMNA Hs.594444 NM_005572,NM_170708, NM_170707 LMO7 Hs.207631 NM_015842, NM_005358 LOC100289511Hs.729250 XM_002347442, XM_002343308, XM_002344795 LOC100302650Hs.729719 LOC282997 Hs.599931 LOC283174 Hs.504370 LOC338799 Hs.524804LOC541471 Hs.652166, Hs.652426, Hs.560805 LOC728392 Hs.104305NM_001162371 LRCH4 Hs.719669, NM_002319 Hs.125742 LRP8 Hs.576154NM_001018054, NM_004631, NM_0033300, NM_017522 LRRN1 Hs.163244 NM_020873LSM14B Hs.105379 NM_144703 LTA Hs.36 NM_001159740, NM_000595 LTBP3Hs.289019 NM_001130144, NM_001164266, NM_021070 LYPD3 Hs.631594NM_014400 MAF Hs.134859 NM_005360, NM_001031804 MAL Hs.80395 NM_022438,NM_022439, NM_0002371, NM_022440 MAMLD1 Hs.20136 NM_005491 MANFHs.436446 NM_006010 MAP2K6 Hs.463978 NM_002758 MAP4K2 Hs.534341NM_004579 MARCKSL1 Hs.75061 NM_023009 MCF2L Hs.170422, NM_001112732,NM_024979 Hs.597691 MDS2 Hs.523369 MED28 Hs.434075, NM_025205 Hs.644788MED6 Hs.497353 NM_005466 MEGF6 Hs.593645 NM_001409 MEOX1 Hs.438NM_001040002, NM_013999, NM_004527 MFGE8 Hs.3745 NM_005928, NM_001114614MINPP1 Hs.121260 NM_004897 MIR1182 MIR155 MIR155HG Hs.697120 MLXIPHs.721711, NM_014938 Hs.437153 MOB1A Hs.602092 NM_018221 MPI Hs.75694NM_002435 MPRIP Hs.462341, NM_201274, NM_015134 Hs.646854 MRPL13Hs.333823 NM_014078 MRPL22 Hs.483924 NM_014180, NM_001014990 MRPL33Hs.515879 NM_145330, NM_004891 MRPL39 Hs.420696 NM_017446, NM_080794MRPL42 Hs.199579 NM_014050, NM_172177, NM_172178 MRPS28 Hs.521124NM_014018 MSC Hs.442619 NM_005098 MSL1 Hs.532786 NM_001012241 MTCH2Hs.269944 NM_014342 MYADM Hs.380906 NM_001020819, NM_001020818,NM_001020821, NM_001020820, NM_138373 MYCBP2 Hs.591221 NM_015057 MYO15BHs.390817 MYOF Hs.602086 NM_013451, NM_133337 MZF1 Hs.399810 NM_198055,NM_003422 NAA50 Hs.596074 NM_025146 NCKAP1 Hs.603732 NM_205842,NM_013436 NDRG2 Hs.525205 NM_201540, NM_201541, NM_201539, NM_201538,NM_201537, NM_201536, NM_201535, NM_016250 NDUFAB1 Hs.189716 NM_005003NDUFAF1 Hs.106529 NM_016013 NDUFV2 Hs.464572 NM_021074 NEDD9 Hs.37982NM_182966, NM_001142393, NM_006403 NEK7 Hs.723303, NM_133494 Hs.24119NELL2 Hs.505326 NM_006159, NM_001145110, NM_001145108, NM_001145109,NM_001145107 NFATC1 Hs.701518, NM_172388, NM_172387, NM_172389,Hs.534074 NM_006162, NM_172390 NIPA1 Hs.511797 NM_144599, NM_001142275NIPAL3 Hs.523442 NM_020448 NLN Hs.247460 NM_020726 NME1 Hs.463456NM_198175, NM_001018138, NM_000269, NM_001018139, NM_002512,NM_001018137, NM_001018136 NME1-NME2 Hs.463456 NM_198175, NM_001018138,NM_001018139, NM_001018137, NM_001018136, NM_000269, NM_002512 NME7Hs.706952 NM_013330, NM_197972 NPEPPS Hs.443837, NM_006310,XM_001725441, Hs.449880 XM_001725426 NQO1 Hs.406515 NM_001025434,NM_001025433, NM_000903 NRCAM Hs.21422 NM_001037132, NM_001037133,NM_005010 NSDHL Hs.57698 NM_015922, NM_001129765 NSMAF Hs.372000NM_003580, NM_001144772 NT5DC3 Hs.48428 NM_001031701 NUBP1 Hs.81469NM_002484 NUCB2 Hs.654599 NM_005013 NUMA1 Hs.325978 NM_006185 NUP153Hs.601591, NM_005124 Hs.718703 OASL Hs.118633 NM_198213, NM_003733 ODC1Hs.467701 NM_002539 OLFM2 Hs.169743 NM_058164 OSBPL7 Hs.463320 NM_145798OTUD7B Hs.98322 NM_020205 P2RY8 Hs.111377 NM_178129 P4HA2 Hs.519568NM_001017974, NM_001017973, NM_001142598, NM_001142599, NM_004199 PAMHs.369430 NM_138766, NM_000919, NM_138822, NM_138821 PAN2 Hs.273397NM_014871, NM_001166279, NM_001127460 PANX2 Hs.440092 NM_001160300,NM_052839 PAPD7 Hs.481542 NM_006999, NM_001171806, NM_001171805 PARK7Hs.419640 NM_007262, NM_001123377 PBX4 Hs.466257 NM_025245 PCIF1Hs.716563 NM_022104 PCSK5 Hs.368542 NM_006200 PDE4A Hs.89901NM_001111308, NM_006202, NM_001111307, NM_001111309 PDIA6 Hs.212102NM_005742 PDK1 Hs.470633 NM_002610 PEA15 Hs.517216 NM_003768 PFKMHs.75160 NM_001166688, NM_001166686, NM_001166687, NM_000289 PGAM1Hs.592599, NM_002629 Hs.632918 PGAM2 Hs.632642 NM_000290 PGAM4 Hs.632822NM_001029891 PHKA2 Hs.54941 NM_000292 PHLPP1 Hs.465337 NM_194449 PHLPP2Hs.709458 NM_015020 PICALM Hs.163893 NM_001008660, NM_007166 PIK3R5Hs.278901 NM_001142633, NM_014308 PIP4K2A Hs.57079 NM_005028 PITPNM2Hs.272759 NM_020845 PLAA Hs.27182 NM_001031689 PLCG1 Hs.268177NM_182811, NM_002660 PLCH2 Hs.170156 NM_014638 PNISR Hs.520287,NM_015491, NM_032870, Hs.644863 POMP Hs.268742 NM_015932 PPFIBP2Hs.655714 NM_003621 PPIL1 Hs.27693 NM_016059 PPP2R1B Hs.584790NM_002716, NM_181699 PPP2R2B Hs.655213 NM_181676, NM_181675, NM_181674,NM_181678, NM_181677, NM_004576, NM_001127381 PPP6R2 Hs.449098,NM_001242898, NM_001242899, Hs.733531, NM_001242900, NM_014678 Hs.740776PPPDE2 Hs.570455 NM_015704 PRDM1 Hs.436023 NM_182907, NM_001198 PRDX4Hs.83383 NM_006406 PREP Hs.436564 NM_002726 PRKAR1B Hs.520851NM_001164760, NM_002735, NM_001164758, NM_001164759, NM_001164762,NM_001164761 PRKCZ Hs.496255 NM_001033581, NM_002744, NM_001033582 PRR5Hs.720401, NM_001017530, NM_181333, Hs.102336 NM_181334, NM_181335,NM_015366, NM_001017526, NM_001017529, NM_001017528 PRSS23 Hs.25338NM_007173 PSMA1 Hs.102798 NM_001143937, NM_148976, NM_002786 PSMB1Hs.352768 NM_002793 PSMC2 Hs.437366 NM_002803 PSMD1 Hs.3887 NM_002807PSMD11 Hs.655396 NM_002815 PSMD14 Hs.567410 NM_005805 PSMD5 Hs.193725NM_005047 PTP4A3 Hs.43666 NM_007079, NM_032611 PTPLA Hs.114062 NM_014241PTPN6 Hs.63489 NM_002831, NM_080548, NM_080549 PTRH2 Hs.12677 NM_016077PUS7 Hs.520619 NM_019042 PYCARD Hs.499094 NM_145182, NM_013258 R3HDM2Hs.443673 NM_014925 RAB1A Hs.310645 NM_004161, NM_015543 RAB21 Hs.524590NM_014999 RAB23 Hs.555016 NM_016277, NM_183227 RAB33A Hs.654356NM_004794 RAB37 Hs.351413 NM_001163990, NM_001163989, NM_175738,NM_001006638 RAB43 Hs.546542, XM_001723593, XM_001720383, Hs.723723XM_001724346, NM_198490, XM_002342369 RABGGTB Hs.78948 NM_004582 RAD50Hs.655835 NM_133482, NM_005732 RAPGEF6 Hs.483329 NM_001164386,NM_001164387, NM_001164388, NM_001164389, NM_001164390, NM_016340 RASA3Hs.593075 NM_007368 RASGRP2 Hs.99491 NM_153819, NM_001098670,NM_001098671 RBBP8 Hs.546282 NM_002894, NM_203292, NM_203291 RBKSHs.11916 NM_022128 REEP5 Hs.429608 NM_005669 RGS1 Hs.75256 NM_002922RGS14 Hs.9347 NM_006480 RHOT2 Hs.513242 NM_138769 RNF19A Hs.292882NM_015435, NM_183419 RNF213 Hs.195642 NM_020914, NM_020954, NM_002343588RNF34 Hs.292804 NM_194271, NM_025126 RPF2 Hs.372265 NM_032194 RPP25Hs.8562 NM_017793 RYBP Hs.7910 NM_012234 S1PR1 Hs.154210 NM_001400 S1PR4Hs.662006 NM_003775 SCML4 Hs.486109 NM_198081 SDHB Hs.465924 NM_003000SDK2 Hs.435719 NM_001144952 SEC24D Hs.189641 NM_014822 SEC31B Hs.18889NM_015490 SELL Hs.728756 NM_000655 SELP Hs.73800 NM_003005 SEPT11Hs.128199 NM_018243 SEPT3 Hs.120483 NM_019106, NM_145733 SEPT9 Hs.440932NM_001113491, NM_001113492, NM_001113493, NM_001113494, NM_001113495,NM_001113496, NM_001293695, NM_001293696, NM_001293697, NM_001293698,NM_006640, SERPINF1 Hs.532768 NM_002615 SERPINF2 Hs.159509 NM_001165920,NM_001165921, NM_000934 SF1 Hs.502829 NM_004630, NM_201995, NM_201997,NM_201998 SFXN1 Hs.369440 NM_022754 SH2B1 Hs.723196 NM_001145797,NM_015503, NM_001145795, NM_001145796, NM_001145812 SHC1 Hs.433795NM_001130040, NM_003029, NM_001130041, NM_183001 SIGIRR Hs.501624NM_021805, NM_001135054, NM_001135053 SIRPG Hs.590883 NM_080816,NM_018556, NM_001039508 SLC16A1 Hs.75231 NM_001166496, NM_003051SLC16A10 Hs.591327 NM_018593 SLC1A4 Hs.654352 NM_003038, NM_001135581SLC24A6 Hs.286194 NM_024959 SLC25A17 Hs.474938 NM_006358 SLC25A32Hs.607819 NM_030780 SLC26A11 Hs.4866 NM_173626, NM_001166348,NM_001166347, NM_001166349 SLC27A2 Hs.720807 NM_003645, NM_001159629SLC2A1 Hs.473721 NM_006516 SLC2A4RG Hs.435126 NM_020062 SLC2A8 Hs.179522NM_014580 SLC35F2 Hs.524014 NM_017515 SLC39A14 Hs.491232 NM_001128431,NM_015359, NM_001135153, NM_001135154 SLC39A8 Hs.288034 NM_022154,NM_001135148, NM_001135147, NM_001135146 SLC40A1 Hs.643005 NM_014585SLC43A3 Hs.99962 NM_199329, NM_017611, NM_014096 SLIRP Hs.655105NM_001267863, NM_001267864, NM_031210 SNPH Hs.713451, NM_014723,NM_001136566 Hs.323833 SNRK Hs.476052 NM_017719, NM_001100594 SNRPGHs.631639, NM_003096, XM_002347904, Hs.654528, NM_001146693,XM_002343626, Hs.516076, XM_001723258 Hs.465167 SNX24 Hs.483200NM_014035 SOAT1 Hs.496383 NM_003101 SORD Hs.878, NM_003104 Hs.633539SOX4 Hs.643910 NM_003107 SP140L Hs.662198 NM_138402 SPATS2L Hs.120323NM_001100424, NM_001100423, NM_001100422, NM_015535 SPG7 Hs.185597NM_003119, NM_199367 SPR Hs.301540 NM_003124 SPSB3 Hs.592080 NM_080861SPTBN1 Hs.503178, NM_003128, NM_178313 Hs.705692 SRGN Hs.1908 NM_002727SRSF5 Hs.632326 NM_001039465, NM_006925 SRXN1 Hs.719997, NM_080725Hs.516830 SSH1 Hs.199763 NM_001161331, NM_001161330, NM_018984 ST8SIA4Hs.308628 NM_175052, NM_005668 STAC Hs.56045 NM_003149 STAT6 Hs.524518NM_003153 STIP1 Hs.337295 NM_006819 STMN3 Hs.639609 NM_015894 STRAPHs.504895 NM_007178 STT3A Hs.504237 NM_152713 STX16 Hs.307913NM_001134772, NM_001134773, NM_003763, NM_001001433 SULT1B1 Hs.129742NM_014465 SUN1 Hs.438072 NM_001171944, NM_001171946, NM_001171945,NM_001130965, NM_025154 SUN2 Hs.517622 NM_015374 SVIL Hs.499209NM_003174, NM_021738 SYT11 Hs.32984 NM_152280 SYTL1 Hs.469175 NM_032872SYTL3 Hs.436977 NM_001009991 TACC3 Hs.104019 NM_006342 TANK Hs.132257NM_004180, NM_133484 TBCC Hs.75064 NM_003192 TBX21 Hs.272409 NM_013351TCEA3 Hs.446354 NM_003196 TCF20 Hs.475018 NM_181492, NM_005650 TCF7Hs.573153 NM_201633, NM_201632, NM_001134851, NM_001134852, NM_213648,NM_003202, NM_201634 TFRC Hs.529618 NM_001128148, NM_003234 THNSL1Hs.645274 NM_024838 TIGIT Hs.421750 NM_173799 TIMD4 Hs.334907NM_001146726, NM_138379 TJP3 Hs.25527 NM_014428 TMC6 Hs.632227NM_001127198, NM_007267 TMC8 Hs.592102 NM_152468 TMCC2 Hs.6360 NM_014858TMED2 Hs.75914, NM_006815 Hs.592682 TMEM110 Hs.556077, NM_198563,NM_205853 Hs.705605 TMEM123 Hs.503709 NM_052932 TMEM165 Hs.479766NM_018475 TMEM220 Hs.462230 NM_001004313 TMEM33 Hs.31082 NM_018126TMEM63A Hs.119387 NM_014698 TMEM66 Hs.521487 NM_016127 TMEM70 Hs.106650NM_017866, NM_001040613 TMEM71 Hs.293842 NM_144649, NM_001145153 TMIGD2Hs.263928 NM_001169126, NM_144615 TNFRSF11A Hs.204044 NM_003839 TNFRSF1BHs.256278 NM_001066 TNFRSF8 Hs.1314 NM_152942, NM_001243 TNFRSF9Hs.654459 NM_001561 TNNT3 Hs.73454 NM_006757, NM_001042782,NM_001042780, NM_001042781 TOP2B Hs.475733 NM_001068 TPM2 Hs.300772NM_213674, NM_003289, NM_001145822 TRAPPC6A Hs.466929 NM_024108 TRIB2Hs.627749, NM_021643 Hs.467751 TRIM22 Hs.501778, NM_006074 Hs.684559TRIP12 Hs.591633 NM_004238 TRMT5 Hs.380159 NM_020810 TSC2 Hs.90303NM_001077183, NM_001114382, NM_000548 TSPAN18 Hs.592575, NM_001031730,NM_130783 Hs.385634 TSPAN32 Hs.271954 NM_139022 TTC4 Hs.720251 NM_004623TTC9 Hs.79170 NM_015351 TTN Hs.134602 NM_133432, NM_133379, NM_133378,NM_133437, NM_003319 TWIST1 Hs.66744 NM_000474 TXK Hs.479669 NM_003328TXN Hs.435136 NM_003329 TXNDC5 Hs.719272, NM_001145549, NM_201280,Hs.150837 NM_030810 UBASH3B Hs.444075 NM_032873 UBE2E2 Hs.595802,NM_152653 Hs.475688 UBE2Z Hs.514297 NM_023079 UCHL3 Hs.162241 NM_006002UCK2 Hs.458360 NM_012474 UHRF1BP1L Hs.620701 NM_001006947, NM_015054USP19 Hs.255596 NM_006677 USP53 Hs.595368, NM_019050 Hs.431081 UXS1Hs.469561 NM_025076 UXT Hs.172791 NM_004182, NM_153477 VDR Hs.524368NM_001017535, NM_000376 VILL Hs.103665 NM_015873 VIPR1 Hs.348500NM_004624 VSIG1 Hs.177164 NM_001170553, NM_182607 VTRNA1-3 WDR12Hs.73291 NM_018256 WNT7A Hs.72290 NM_004625 WRB Hs.198308 NM_001146218,NM_004627 XAF1 Hs.441975 NM_017523, NM_199139 YPEL3 Hs.513491 NM_031477,NM_001145524 YWHAE Hs.591239, NM_006761 Hs.513851 YWHAG Hs.520974NM_012479 ZBTB22 Hs.206770 NM_005453, NM_001145338 ZBTB38 Hs.723156NM_001080412 ZC3H12C Hs.376289 NM_033390 ZDHHC16 Hs.76662 NM_032327,NM_198046, NM_198045, NM_198044, NM_198043 ZFP36L2 Hs.503093 NM_006887ZGPAT Hs.590868 NM_181485, NM_001083113, NM_032527 ZMAT1 Hs.496512NM_032441, NM_001011657 ZNF193 Hs.100921 NM_006299 ZNF238 Hs.69997NM_205768, NM_006352 ZNF282 Hs.657701 NM_003575 ZNF331 Hs.185674NM_018555, NM_001079907, NM_001079906 ZNF506 Hs.351906 NM_001145404,NM_001099269 ZNF542 Hs.467326 ZNF673 Hs.632800 NM_017776, NM_001129900,NM_001129898, NM_001129899 ZNF688 Hs.301463, NM_152458, NM_145271,Hs.513509 NM_001024683 ZNF710 Hs.459311 NM_198526 ZNF83 Hs.710125,NM_018300, NM_001105550, Hs.665751, NM_001105552, NM_001105551,Hs.467210, NM_001105554, NM_001105553, Hs.659798 NM_001105549 ZSWIM1Hs.517075 NM_080603

The most significant genes in Table 7A were defined as those with 1) anabsolute fold change between ALL CRs/CLL CRs and CLL NRs of greater than2 and 2) a p-value on the correlation of response and expression of lessthan 0.01. Thirty-four genes, listed in Table 7B below, met thiscriteria and the expression of these genes were measured on fouradditional platforms to compare and validate the findings from RNAseq.The four platforms were OpenArray, Fluidigm, Nanostring, and qPCR.Results from this cross-platform comparison experiment confirmed theresults and conclusions described herein. Table 7B also indicateswhether each gene is upregulated in complete responders (CR) relative tonon-responders, or upregulated in non-responders (NR) relative tocomplete responders. An exemplary publication disclosing the sequence ofeach gene is also given in Table 7B, and each publication isincorporated by reference in its entirety, including all nucleic acidand protein sequences therein.

TABLE 7B Table 7B. Selected genes from Table 7A Upregulated Gene UnigeneAccession No. Exemplary publication in CR or NR ALS2CL Hs.517937NM_147129, Jouan et al., Behav Brain Funct CR NM_182775 9, 9 (2013) AQP3Hs.234642 NM_004925 Xie et al., Arch. Dermatol. Res. CR 305 (5), 397-406(2013) C16orf74 Hs.461655 NM_206967 Kim et al., PLoS ONE 5 (12), CRE15260 (2010) CCL17 Hs.546294 NM_002987 Lee et al, Pediatr. Res. 74 (5),NR 545-551 (2013) CD248 Hs.195727 NM_020404 Kontsekova et al., Int. J.Oncol. CR 41 (4), 1365-1372 (2012) CSF2 Hs.1349 NM_000758 Sawada et al.,J. Exp. Med. 211 NR (2), 263-280 (February 2014) DHRS2 Hs.272499NM_182908, Prunotto et al., J Proteomics NR NM_005794 82, 193-229 (2013)DPEP2 Hs.372633 NM_022355 Willer et al., Nat. Genet. 40 CR (2), 161-169(2008) EPAS1 Hs.468410 NM_001430 Mathew et al., Proc. Natl. NR Acad.Sci. U.S.A. 111 (1), 291- 296 (January 2014) EPHA4 Hs.371218 NM_004438Xu et al., Proc. Natl. Acad. Sci. CR U.S.A. 110 (36), 14634-14639 (2013)FAIM3 Hs.723317, NM_001142472, Murakami et al., J. Immunol. CR Hs.58831NM_001142473, 189 (2), 587-597 (2012) NM_005449 FAM134B Hs.711125NM_001034850, Murphy et al., J. Neurol. CR NM_019000 Neurosurg.Psychiatr. 83 (1), 119-120 (2012) GPA33 Hs.651244 NM_005814 Deng et al.PLoS ONE 8 (11), CR E79629 (2013) IL13 Hs.845 NM_002188 Jiang et al.,Am. J. Physiol. NR Endocrinol. Metab. 305 (11), E1359-E1366 (2013) IL3Hs.694 NM_000588 Miyake et al., Cytokine 64 (1), NR 86-89 (2013) IL9Hs.960 NM_000590 Jabeen et al., J. Clin. Invest. NR 123 (11), 4641-4653(2013) KRT72 Hs.662013 NM_080747, Principe et al., Proteomics 13 CRNM_001146226, (10-11), 1667-1671 (2013) NM_001146225 KRT73 Hs.55410NM_175068 De Mateo et al., Proteomics 11 CR (13), 2714-2726 (2011) LTAHs.36 NM_001159740, Stuart et al., Twin Res Hum NR NM_000595 Genet 16(6), 1079-1086 (2013) MCF2L Hs.170422, NM_001112732, Valdes et al., Ann.Rheum. CR Hs.597691 NM_024979 Dis. 71(9), 1537-1540 (2012) MDS2Hs.523369 Meyer et al., PLoS Genet. 6 (8) CR (2010) MEGF6 Hs.593645NM_001409 Nakayama et al. Genomics 51 CR (1), 27-34 (1998) MIR155 Weberet al., FEBS J. 272 (1), NR 59-73 (2005) PPFIBP2 Hs.655714 NM_003621Bohm et al., Oncol. Rep. 28 CR (2), 429-438 (2012) SCML4 Hs.486109NM_198081 Vieira et al., Genet. Med. 10 CR (9), 668-674 (2008) SDK2Hs.435719 NM_001144952 Otowa et al., J. Hum. Genet. 54 CR (2), 122-126(2009) SPR Hs.301540 NM_003124 Yang et al., J. Biol. Chem. 288 NR (26),19221-19237 (2013) SULT1B1 Hs.129742 NM_014465 Ross et al., Nat. Genet.41 (12), CR 1345-1349 (2009) TCF7 Hs.573153 NM_201633, Nikuseva-Marticet al., Pathol. CR NM_201632, Oncol. Res. 19 (3), 545-551 NM_001134851,(2013) NM_001134852, NM_213648, NM_003202, NM_201634 TNFRSF8 Hs.1314NM_152942, Yao et al., Am. J. Surg. Pathol. NR NM_001243 37 (9),1407-1412 (2013) TSPAN18 Hs.592575, NM_001031730, Yuan et al., PLoS ONE8 (3), CR Hs.385634 NM_130783 E58785 (2013) TWIST1 Hs.66744 NM_000474Zhou et al., J. Exp. Clin. NR Cancer Res. 33, 12 (January 2014) VIPR1Hs.348500 NM_004624 Bono et al., Cancer Cell 23 (4), CR 477-488 (2013)VSIG1 Hs.177164 NM_001170553, Chen et al., J Surg Oncol 106 CR NM_182607(3), 286-293 (2012)

Cell surface markers differentiating memory T cell subsets that aredescribed in Maus et al. (ANNU. REV. IMMUNOL. 2014) and were notincluded in the Gattinoni gene sets were also evaluated. Among otherthings, KLRG1 was identified as a gene whose expression increases inapheresis samples from ALL→CLL CR→CLL PR→CLL NR. KLRG1 expression valuespredict patient response to CTL019 therapy. At least CD57, CD27, CD122,and CD62L were identified as biomarkers of response in the productsamples. Among other things CD57, CD27, CD122, and CD62L expressionvalues predict patient response to CTL019 therapy.

In an embodiment, a complete responder (CR) gene signature comprises oneor more biomarker profiles described in Table 9.

TABLE 9 Exemplary biomarker profile of a complete responder to CAR19therapy Table 9: Exemplary biomarker profile of a complete responder toCAR19 therapy CD27+ PD1− CD8+ LAG3− TIM3− KLRG1− Exemplary CR cell-typesResting T effector cells (T_(EM)) Resting T_(REG) Nave CD4+ Unstimulatedmemory T cells (T_(SCM)) Early memory T cells

In an embodiment, a non-responder (NR) gene signature comprises one ormore biomarker profiles described in Table 10.

TABLE 10 Exemplary biomarker profile of a non-responder to CAR19 therapyTable 10: Exemplary biomarker profile of a non-responder to CAR19therapy PD1+ CD27− LAG3+ TIM3+ KLRG1+ Exemplary NR cell-types ActivatedT effector cells (T_(EM)) Activated T_(REG) Activated T_(H1) RestingT_(H2) Stimulated memory T cells (T_(SCM)) Late memory T cells

Based on the biological understanding, combinations of genes fromunbiased feature selection, gene sets, and selected genes of interestcould be used to further differentiate NR's, PR's, and CR's.

The previously described work was expanded upon in a study of 35 CLLsubject samples. This group of 35 subjects includes the 21 CLL subjectsin the previous study, for a total of 5 CRs, 9 PRs, and 21 NRs. In thisstudy manufactured CD19 CAR-expressing cell product samples werecollected and cultured overnight with control beads. Novel genesignatures based on mRNA expression levels have been identified thatpredict patient response. The gene lists for the CR vs NR comparison(N=185) is tabulated in Table 18.

TABLE 18 Gene lists for the CR vs NR comparison Gene Unigene AccessionNo. FDR ABCB1 Hs.489033 NM_000927 0.0492 ACSL1 Hs.406678 NM_001995,NM_001286711, NM_001286708, 0.0362 NM_001286710, NM_001286712 ADAM12NM_003474, NM_001288973, NM_021641, NM_001288974, 0.0089 NM_001288975ADAM23 NM_003812 0.0185 ADCY1 Hs.192215 NM_001281768, NM_021116 0.0203AFAP1 Hs.529369 NM_198595, NM_001134647 0.0884 AGRN Hs.273330 NM_198576,NM_001305275 0.0212 ANKRD33B Hs.26039 NM_001164440 0.0097 APLP2Hs.370247 NM_001142277, NM_001642, NM_001142278, 0.0429 NM_001142276,NM_001243299 AQPEP 0.0615 ARHGAP32 Hs.440379 NM_001142685, NM_0147150.0675 ART3 Hs.731997 NM_001130017, NM_001130016, NM_001130016, 0.0890NM_001179 ATN1 Hs.143766 NM_001007026, NM_001940 0.0675 ATP9A Hs.649234NM_006045 0.0029 B4GALNT1 Hs.159481 NM_001478, NM_001276468,NM_001276469 0.0104 B4GALT6 NM_004775 0.0965 C1orf198 Hs.520494NM_032800, NM_001136494, NM_001136495 0.0047 C21orf63 0.0029 C5orf390.0047 C9orf142 NM_183241 0.0870 CACNB2 Hs.59093 NM_201596, NM_201593,NM_201597, NM_201571, 0.0615 NM_001167945, NM_201572, NM_000724,NM_201590, NM_201570 CAMK2G NM_001222, NM_172173, NM_172169, NM_172170,0.0675 NM_001204492, NM_172171 CAMSAP2 Hs.23585 NM_203459, NM_001297708,NM_001297707 0.0797 CCDC74A Hs.351461 NM_138770, NM_001258304,NM_001258306, 0.0492 NM_001258305 CCL22 Hs.534347 NM_002990 0.0666 CCL5NM_002985, NM_001278736 0.0615 CD109 Hs.399891 NM_133493, NM_001159588,NM_001159587 0.0423 CD200 NM_001004196, NM_005944 0.0299 CD27 Hs.355307NM_001242 0.0225 CD52 NM_001803 0.0172 CDKN1A Hs.370771 NM_000389,NM_001220778, NM_001220777, NM_078467 0.0450 CERS6 Hs.743222 NM_203463,NM_001256126 0.0384 CHST2 Hs.8786 NM_004267 0.0063 CNTNAP2 Hs.655684NM_014141 0.0872 CPA5 Hs.567642 NM_001127442, NM_080385, NM_0011274410.0433 CPM Hs.654387 NM_001005502, NM_198320, NM_001874, NM_001005502,0.0466 NM_198320, NM_001874 CR1 NM_000573, NM_000651 0.0891 CTNNA1Hs.445981, NM_001903, NM_001290310, NM_001290309, 0.0148 Hs.740112NM_001290307, NM_001290312 CXCL9 Hs.77367 NM_002416 0.0299 CXCR5NM_001716 0.0939 DBN1 NM_004395, NM_080881 0.0492 DEPDC7 Hs.280990NM_001077242, NM_139160 0.0256 DIRC3 0.0890 DLG2 Hs.367656 NM_001364,NM_001142702, NM_001142699, 0.0085 NM_001142700, NM_001300983,NM_001206769 DNAJC12 Hs.260720 NM_021800, NM_201262 0.0891 DRAM1Hs.525634 NM_018370 0.0376 DSG2 Hs.412597 NM_001943 0.0384 DUSP4NM_001394, NM_057158 0.0148 EBI3 NM_005755 0.0063 EEF1A2 Hs.433839NM_001958 0.0497 EEF1DP3 0.0939 EHD4 NM_139265 0.0415 EMP1 NM_0014230.0541 ENPP2 Hs.190977 NM_006209, NM_001130863, NM_001040092 0.0870EPAS1 Hs.468410 NM_001430 0.0149 ERP29 NM_006817, NM_001034025 0.0615EVC Hs.646899 NM_001306090, NM_153717, NM_001306092 0.0666 EVI5Hs.594434 NM_005665, NM_001308248 0.0373 FADS2 NM_001281501,NM_001281502, NM_004265 0.0764 FAM134B Hs.481704 NM_019000, NM_0010348500.0452 FAM40B 0.0148 FAM65C Hs.372578 NM_001290268, NM_080829 0.0615FASN Hs.83190 NM_004104 0.0884 FKBP11 NM_001143781, NM_016594,NM_001143782 0.0407 FLT1 Hs.594454 NM_002019, NM_001160030,NM_001159920, 0.0699 NM_001160031 FLT3LG NM_001204502, NM_001459,NM_001278637, 0.0408 NM_001278638, NM_001204503 FOXP1 NM_032682,NM_001244816, NM_001244815, 0.0615 NM_001244814, NM_001244808,NM_001244812, NM_001012505, NM_001244813, NM_001244810 FSCN1 Hs.118400NM_003088 0.0694 GAS8 Hs.431792, NM_001481, NM_001286209, NM_001286205,0.0811 Hs.739124 NM_001286208 GEM Hs.654463 NM_181702, NM_005261 0.0275GNA12 Hs.487341 NM_007353, NM_001282441, NM_001282440 0.0360 GPR560.0122 GZMA NM_006144 0.0805 HCST NM_014266, NM_001007469 0.0212 HDCHs.1481 NM_002112, NM_001306146 0.0890 HSH2D Hs.631617 NM_032855 0.0243IL1A NM_000575 0.0148 IL1RN NM_173843, NM_173841, NM_000577, NM_1738420.0615 IL26 Hs.272350 NM_018402 0.0718 ILDR2 Hs.133153, NM_199351 0.0860Hs.730291 KLRB1 Hs.169824 NM_002258 0.0074 KLRC3 Hs.654362 NM_002261,NM_007333 0.0035 LHFP Hs.507798 NM_005780 0.0053 LIFR Hs.133421,NM_002310, NM_001127671 0.0362 Hs.616721 LINC00476 0.0299 LMCD1NM_014583, NM_001278235, NM_001278233, 0.0423 NM_001278234 LMNAHs.594444 NM_001282625, NM_005572, NM_170707, NM_001282626, 0.0677NM_001257374, NM_001282624, NM_170708 LOC347411 0.0407 LOC619207 0.0407LRIG3 NM_001136051, NM_153377 0.0746 LRP1B Hs.656461 NM_018557 0.0615LRRC4C Hs.745123 NM_001258419, NM_020929 0.0733 LY9 Hs.403857NM_001261456, NM_001261457, NM_002348, 0.0615 NM_001033667 MAST2Hs.319481 NM_015112 0.0959 MGAT4A Hs.177576 NM_001160154, NM_0122140.0085 MOB1B Hs.691454 NM_173468, NM_001244766 0.0441 MRPL54 NM_1722510.0910 MYOF Hs.602086 NM_133337, NM_013451 0.0615 NAB2 Hs.159223NM_005967 0.0373 NCDN Hs.121870 NM_001014841, NM_001014839, NM_0142840.0876 NCKAP1 Hs.603732 NM_013436, NM_205842 0.0299 NCR3 NM_147130,NM_001145466, NM_001145467 0.0595 NDUFA12 Hs.674965 NM_018838,NM_001258338 0.0936 NEDD4L Hs.185677 NM_001243960, NM_001144967,NM_015277, 0.0333 NM_001144971, NM_001144968, NM_001144969,NM_001144970, NM_001144966, NM_001144964, NM_001144965 NEURL3 Hs.149219NM_001285485, NM_001285486 0.0821 NINL Hs.631508 NM_025176 0.0709 NOSIPHs.7236 NM_001270960, NM_015953 0.0001 NRP2 Hs.471200 NM_201266,NM_201264, NM_201267, NM_003872, 0.0926 NM_018534, NM_201279 OSMRHs.120658 NM_001168355, NM_003999 0.0432 PANX2 Hs.440092 NM_001160300,NM_052839 0.0376 PCBP3 Hs.736936 NM_020528, NM_001130141 0.0089 PHKA1Hs.201379 NM_002637, NM_001122670, NM_001172436 0.0130 PITPNC1 Hs.591185NM_181671, NM_012417 0.0373 PLXNB2 Hs.3989 NM_012401 0.0148 PLXNB3Hs.632833 NM_005393, NM_001163257 0.0981 PMCH NM_002674 0.0383 POU2AF1Hs.654525, NM_006235 0.0376 Hs.733573, Hs.739353 PPARG Hs.162646NM_138712, NM_005037, NM_138711, NM_015869 0.0821 PPCDC Hs.458922,NM_021823, NM_001301103, NM_001301101, 0.0224 Hs.640486 NM_001301102,NM_001301104, NM_001301105 PRDM1 Hs.436023 NM_001198, NM_182907 0.0376PRKCDBP NM_145040 0.0615 PRR5 NM_015366, NM_001198721, NM_181333,NM_001017528, 0.0384 NM_001017530, NM_001017529 PSEN2 Hs.25363NM_000447, NM_012486 0.0299 PTPN6 Hs.63489 NM_080548, NM_002831,NM_080549 0.0733 PTPRCAP NM_005608 0.0521 PTPRD Hs.446083 NM_002839,NM_130391, NM_001171025, NM_130393, 0.0141 NM_001040712, NM_130392 PVRNM_006505, NM_001135769, NM_001135768, 0.0149 NM_001135770 RABL3Hs.444360 NM_173825 0.0733 RBMY1E NM_001006118 0.0089 RGL1 Hs.497148NM_001297669, NM_015149, NM_001297670, 0.0891 NM_001297671, NM_001297672RNASE4 NM_001282193, NM_001282192, NM_002937, NM_194431 0.0860 RORCHs.256022 NM_001001523, NM_005060 0.0027 RPS28 NM_001031 0.0763 S100A4NM_019554, NM_002961 0.0047 SCARB1 NM_001082959, NM_005505 0.0327 SCDHs.558396 NM_005063 0.0870 SCML4 NM_001286408, NM_001286409, NM_1980810.0860 SDC1 Hs.224607 NM_002997, NM_001006946 0.0299 SDK2 Hs.435719NM_001144952 0.0981 SEPT3 Hs.120483 NM_145733, NM_019106 0.0582SEPT5-GP1BB 0.0107 SGPP2 Hs.591604 NM_152386 0.0347 SH2B2 Hs.489448NM_020979 0.0373 Hs.479116, NM_018986 0.0205 SH3TC1 Hs.630085 SKAP1Hs.316931 NM_003726, NM_001075099 0.0661 SLC13A3 Hs.655498 NM_022829,NM_001193340, NM_001193339, 0.0254 NM_001193342, NM_001011554 SLC22A17Hs.373498 NM_001289050, NM_016609, NM_020372 0.0944 SLC27A2 Hs.11729NM_003645, NM_001159629 0.0811 SLC29A1 Hs.25450 NM_001304463,NM_001078175, NM_001078177, 0.0150 NM_001304465, NM_001304466,NM_001304462 SLC41A2 NM_032148 0.0936 SLC43A3 Hs.99962 NM_014096,NM_001278201, NM_199329, NM_017611, 0.0423 NM_001278206 SLC4A10Hs.333958 NM_022058, NM_001178015, NM_001178016 0.0376 SOAT2 Hs.656544NM_003578 0.0130 SORCS3 Hs.671950 NM_014978 0.0876 SPIRE1 Hs.515283NM_001128626, NM_020148, NM_001128627 0.0615 SPNS3 NM_182538 0.0001SPOCK1 Hs.582184, NM_004598 0.0718 Hs.596136 SRCIN1 Hs.448872 NM_0252480.0661 SSBP3 Hs.733025 NM_145716, NM_001009955, NM_018070 0.0376 STX8NM_004853 0.0937 SULT2B1 Hs.369331 NM_177973, NM_004605 0.0150 TERTHs.492203 NM_198253, NM_001193376, NM_005424 0.0765 TIE1 Hs.78824NM_005424, NM_001253357 0.0931 TLE4 NM_007005, NM_001282760,NM_001282748, 0.0891 NM_001282749, NM_001282753 TMOD1 Hs.404289NM_003275, NM_001166116 0.0308 TNFRSF19 NM_148957, NM_018647,NM_001204458, NM_001204459 0.0224 TNFRSF4 Hs.129780 NM_003327 0.0884TOB1 Hs.744946 NM_005749, NM_001243885, NM_001243877 0.0945 TOX2Hs.26608 NM_001098797, NM_001098796, NM_032883, 0.0347 NM_001098798TRIB2 Hs.467751 NM_021643 0.0666 TSKU Hs.8361 NM_015516, NM_0012582100.0027 TSPAN13 NM_014399 0.0376 TTBK1 Hs.485436 NM_032538 0.0027 TTC39CHs.733420 NM_153211, NM_001243425, NM_001135993, 0.0172 NM_001292030TUBB6 Hs.193491, NM_001303524, NM_032525, NM_001303529, 0.0271 Hs.744066NM_001303526, NM_001303525 uc001acl 0.0212 uc004aex 0.0299 uc010eif0.0243 uc021oxp 0.0595 USP44 NM_032147, NM_001042403, NM_0012783930.0666 XYLT1 Hs.22907 NM_022166 0.0890 ZBTB20 NM_001164343,NM_001164347, NM_001164345, 0.0148 NM_001164342, NM_015642,NM_001164344, NM_001164346 ZBTB32 Hs.99430, NM_014383 0.0384 Hs.736841ZNF219 Hs.250493 NM_016423, NM_001102454, NM_001101672 0.0860 ZNF683Hs.353208 NM_001114759, NM_173574, NM_001307925 0.0205

Gene set analyses were performed to predict patient response to CD19CAR-expressing cell therapy (e.g., CTL019). Gene set analysis wasperformed on gene sets described in Example 1, and with gene sets fromthree additional datasets described in Example 2 (Szabo et al., Abbas etal., and Gattinoni et al.). Each gene set was evaluated to determine itsassociation with subject response (i.e. CR, PR, or NR) as described inExample 2. Gene sets found to be significantly altered and predictive ofpatient response to CD19 CAR-expressing cell therapy (e.g., CTL019) arelisted in Table 19.

TABLE 19 Gene sets predictive of patient response to CAR therapy GeneSet Source CRs NRs Treg vs Teff 0 h Szabo Teff 0 h Treg 0 h Treg vs Teff16 h Szabo Teff 16 h Treg 16 h Teff 16 h vs 0 h Szabo Teff 0 h Teff 16 hTreg 16 h vs 0 h Szabo Treg 0 h Treg 16 h Nave CD4 vs 12 h act Th2 AbbasNave CD4 Th2 Nave CD4 vs 48 h act Th2 Abbas Nave CD4 Th2 Nave CD4 vs 12h act Th1 Abbas Nave CD4 Th1 Unstim vs stim memory Abbas UnstimulatedStimulated Progressively down Gattinoni Early stage Late stage

Significant gene sets from the analyses above were refined to a subsetof genes within the gene sets that are significantly differentiallyexpressed between the CRs and NRs. An exemplary listing of genes thatwere significantly differentially expressed are listed in Table 20.Table 20 is an exemplary list of biomarkers whose expression valuespredict patient response to CTL019 therapy. Table 20 can be furtherrefined into a smaller list of high confidence biomarkers by setting astricter FDR. For instance, using a FDR of 0.10 will results in a listof 265 genes and a FDR of 0.01 will result in a list of 27 genes.

TABLE 20 Exemplary biomarkers for predicting patient response to CARtherapy Gene Unigene Accession No. FDR ABCB1 Hs.489033 NM_000927 0.0147ABTB1 Hs.107812 NM_172027, NM_032548 0.156 ACACA NM_198834, NM_198837,NM_198836, 0.226 NM_198839, NM_198838 ACSL1 Hs.406678 NM_001995,NM_001286711, NM_001286708, 0.0211 NM_001286710, NM_001286712 ACSS2Hs.517034 NM_001242393, NM_018677, NM_001076552 0.189 ACTN1 Hs.509765NM_001102, NM_001130004, NM_001130005 0.137 ADAM12 NM_003474,NM_001288973, NM_021641, 0.00259 NM_001288974, NM_001288975 ADAM7Hs.116147 NM_003817 0.249 ADD3 NM_019903, NM_016824, NM_001121 0.243ADH7 Hs.389 NM_001166504, NM_000673 0.179 AES Hs.515053 NM_198969,NM_001130, NM_198970 0.0307 AGRN Hs.273330 NM_198576, NM_0013052750.00795 AHCYL1 Hs.743973 NM_006621, NM_001242675, NM_001242676, 0.24NM_001242673, NM_001242674 AHI1 NM_001134830, NM_017651, NM_001134831,0.106 NM_001134832 AIM2 NM_004833 0.224 AK5 Hs.559718 NM_174858,NM_012093 0.21 AKR1C3 NM_003739, NM_001253908 0.212 ALDH18A1 Hs.500645NM_002860, NM_001017423 0.234 ALG5 NM_013338, NM_001142364 0.171 ALOX5APNM_001629, NM_001204406 0.0878 ALPP Hs.284255 NM_001632 0.218 ANAPC13Hs.106909 NM_001242374, NM_015391, NM_001242375 0.246 ANKRD10 NM_017664,NM_001286721 0.229 ANKRD6 NM_001242813, NM_001242809, NM_014942, 0.0842NM_001242811, NM_001242814 ANXA2P3 0.219 AP1G2 Hs.343244, NM_001282475,NM_001282474, NM_003917 0.0756 Hs.740123 APOA2 NM_001643 0.0265 APOBEC3CNM_014508 0.11 APP Hs.434980 NM_000484, NM_001136129, NM_201414, 0.129NM_001204303, NM_201413, NM_001204302, NM_001136016, NM_001136130,NM_001204301, NM_001136131 AQP3 Hs.234642 NM_004925 0.0645 ARFRP1Hs.389277, NM_001267549, NM_001134758, NM_001267544, 0.112 Hs.661969NM_001267546, NM_001267545, NM_001267547, NM_003224, NM_001267548ARHGEF18 Hs.465761 NM_015318, NM_001130955 0.207 ARID5A NM_212481 0.184ARL4C Hs.111554, NM_001282431, NM_005737 0.091 Hs.730678 ARNTL2NM_001248003, NM_001248005, NM_001248002, 0.0709 NM_001248004, NM_020183ARRDC2 Hs.515249 NM_001025604, NM_001286826, NM_015683 0.248 ATF7IP2Hs.513343, NM_024997, NM_001256160 0.0661 Hs.742019 ATP6V1B2 Hs.295917NM_001693 0.171 ATP8A2 NM_016529 0.222 AURKAIP1 NM_017900, NM_001127230,NM_001127229 0.207 AUTS2 Hs.21631 NM_001127231, NM_015570, NM_0011272320.158 B4GALT7 Hs.455109 NM_007255 0.115 BACH2 Hs.269764 NM_021813,NM_001170794 0.115 BANP Hs.461705, NM_001173541, NM_079837,NM_001173540, 0.236 Hs.690969 NM_017869, NM_001173543, NM_001173539,NM_001173542 BARD1 NM_000465, NM_001282549, NM_001282543, 0.232NM_001282548, NM_001282545 BASP1 Hs.201641 NM_006317, NM_001271606 0.137BCL11B Hs.709690 NM_001282237, NM_138576, NM_001282238, 0.134 NM_022898BCOR NM_017745, NM_001123384, NM_001123385, 0.22 NM_001123383 BEX4Hs.184736 NM_001080425, NM_001127688 0.218 BFSP1 Hs.129702 NM_001195,NM_001161705, NM_001278607, 0.186 NM_001278606, NM_001278608 BHLHE40NM_003670 0.224 BIN1 Hs.193163 NM_139344, NM_139348, NM_139351, 0.192NM_139346, NM_139347, NM_139349, NM_139345, NM_004305, NM_139343,NM_139350 BIN2 Hs.14770 NM_001290008, NM_001290009, NM_016293, 0.094NM_001290007 BIRC3 NM_182962, NM_001165 0.127 BUB1 Hs.469649NM_001278617, NM_004336, NM_001278616 0.141 C11orf21 Hs.559181NM_001142946 0.0679 C11orf48 0.103 C11orf67 0.227 C11orf82 0.196C14orf49 0.217 C16orf45 Hs.738182 NM_033201, NM_001142469 0.181 C16orf74NM_206967 0.197 C17orf53 Hs.437059 NM_024032, NM_001171251 0.185C17orf66 0.229 C1orf162 Hs.288010 NM_174896, NM_001300834 0.135 C1orf54NM_001301040, NM_001301039, NM_024579, 0.212 NM_001301042 C20orf1110.0568 C20orf112 0.102 C2orf28 0.24 C2orf89 0.159 C5orf30 Hs.482976NM_033211 0.207 C5orf39 0.00226 C7orf10 0.162 C7orf59 0.0455 C9orf230.0943 CA6 NM_001215, NM_001270500, NM_001270501 0.0798 CAMK1 NM_0036560.215 CAMK2G NM_001222, NM_172173, NM_172169, 0.0175 NM_172170,NM_001204492, NM_172171 CAMK4 NM_001744 0.0524 CAPG Hs.687978 NM_001747,NM_001256140, NM_001256139 0.0735 CAPS NM_080590, NM_004058 0.0524 CARM1NM_199141 0.162 CBLB Hs.430589 NM_170662 0.061 CCDC47 Hs.202011NM_020198 0.104 CCDC56 0.0674 CCL20 Hs.75498 NM_001130046, NM_0045910.249 CCL4L1 0.0434 CCL5 NM_002985, NM_001278736 0.015 CCNB1 Hs.23960NM_031966 0.195 CCND3 NM_001760, NM_001287427, NM_001136126, 0.0618NM_001136017, NM_001136125, NM_001287434 CCR6 NM_031409, NM_0043670.0767 CD109 Hs.399891 NM_133493, NM_001159588, NM_001159587 0.013 CD200NM_001004196, NM_005944 0.00952 CD22 Hs.579691, NM_001185099, NM_001771,NM_001185100, 0.124 Hs.716252 NM_001278417, NM_001185101 CD244 Hs.157872NM_016382, NM_001166663, NM_001166664 0.0605 CD248 Hs.195727 NM_0204040.0548 CD3D NM_000732, NM_001040651 0.0455 CD4 NM_000616 0.166 CD5Hs.58685 NM_014207 0.0926 CD55 NM_000574, NM_001300903, NM_001300904,0.139 NM_001300902, NM_001114752 CD68 Hs.647419 NM_001251, NM_0010400590.144 CD80 Hs.838 NM_005191 0.061 CDC14A Hs.127411 NM_033312, NM_033313,NM_003672 0.149 CDC25B Hs.153752 NM_001287519, NM_001287520, NM_021873,0.136 NM_004358, NM_001287522, NM_021872, NM_001287518, NM_001287516,NM_001287517 CDC42BPB Hs.654634 NM_006035 0.0835 CDC42EP3 Hs.369574,NM_006449, NM_001270437, NM_001270438, 0.126 Hs.689535 NM_001270436 CDC6NM_001254 0.201 CDKN1A Hs.370771 NM_000389, NM_001220778, NM_001220777,0.013 NM_078467 CDKN2D Hs.435051 NM_079421, NM_001800 0.218 CDT1NM_030928 0.195 CECR1 Hs.170310 NM_177405, NM_001282228, NM_001282227,0.147 NM_001282229, NM_001282226, NM_001282225 CEMP1 NM_001048212 0.179CEP55 Hs.14559 NM_001127182, NM_018131 0.241 CFH Hs.363396 NM_000186,NM_001014975 0.158 CFHR2 NM_005666 0.074 CGREF1 Hs.159525 NM_001166240,NM_006569, NM_001166239 0.201 CHEK1 Hs.24529 NM_001114121, NM_001274,NM_001244846, 0.136 NM_001114122 CHL1 Hs.148909, NM_006614,NM_001253387, NM_001253388 0.226 Hs.731409 CHMP7 Hs.5019 NM_1522720.0594 CHST11 Hs.17569 NM_001173982, NM_018413 0.149 CHST12 Hs.744987NM_001243794, NM_001243795, NM_018641 0.192 CHST2 Hs.8786 NM_0042670.00226 CHSY1 Hs.110488, NM_014918 0.11 Hs.734921 CLCA2 Hs.241551NM_006536 0.172 CMAHP 0.0642 CNPY3 NM_006586 0.0605 COL18A1 Hs.517356NM_130445, NM_030582, NM_130444 0.189 COL6A1 Hs.474053 NM_001848 0.201CORO1C Hs.330384 NM_014325, NM_001276471, NM_001105237 0.0566 COX4I1NM_001861 0.222 CRADD Hs.591016, NM_003805 0.144 Hs.719191 CRKL Hs.5613NM_005207 0.201 CSGALNACT1 Hs.613729 NM_001130518, NM_018371 0.0589CSNK2A1 NM_001895, NM_177559, NM_177560 0.171 CSTB NM_000100 0.0477CSTF2 NM_001325, NM_001306206 0.158 CTDSP1 Hs.444468 NM_001206878,NM_182642, NM_021198 0.195 CTNNA1 Hs.445981, NM_001903, NM_001290310,NM_001290309, 0.00562 Hs.740112 NM_001290307, NM_001290312 CTNNA2Hs.167368 NM_004389, NM_001282598, NM_001164883, 0.145 NM_001282597,NM_001282600, NM_001282599 CTNNAL1 NM_001286974, NM_003798 0.124CTNNBIP1 Hs.463759 NM_020248, NM_001012329 0.091 CTNND2 Hs.314543NM_001332, NM_001288717, NM_001288715, 0.226 NM_001288716 CTSF Hs.11590NM_003793 0.224 CTSW Hs.416848 NM_001335 0.0929 CTTN Hs.596164NM_138565, NM_005231, NM_001184740 0.0642 CUX1 Hs.191482 NM_181500,NM_001202546, NM_001202544, 0.137 NM_001202543, NM_001202545, NM_181552,NM_001913 CXCL13 Hs.100431 NM_006419 0.192 CYB561 Hs.355264NM_001017917, NM_001915, NM_001017916 0.208 CYFIP1 Hs.26704 NM_014608,NM_001033028, NM_001287810 0.192 D4S234E 0.224 DAB1 Hs.477370 NM_0210800.0455 DAXX NM_001141969, NM_001254717, NM_001141970, 0.192 NM_001350DBN1 NM_004395, NM_080881 0.0127 DENND2D NM_024901, NM_001271833 0.0882DENND3 Hs.18166 NM_014957 0.223 DEPDC7 Hs.280990 NM_001077242, NM_1391600.00865 DGKD Hs.471675 NM_152879, NM_003648 0.0524 DGKI Hs.737768NM_004717 0.0596 DHCR24 Hs.498727 NM_014762 0.0843 DIXDC1 Hs.655626NM_001278542, NM_001037954, NM_033425 0.224 DLG2 Hs.367656 NM_001364,NM_001142702, NM_001142699, 0.00226 NM_001142700, NM_001300983,NM_001206769 DMRT1 Hs.98586 NM_021951 0.0589 DNAJB5 Hs.237506NM_001135004, NM_012266, NM_001135005 0.0843 DNAJC6 Hs.647643 NM_014787,NM_001256864, NM_001256865 0.219 DNM1 Hs.522413 NM_004408, NM_001005336,NM_001288737, 0.244 NM_001288738, NM_001288739 DOCK7 Hs.744927NM_033407, NM_001272000, NM_001272002, 0.061 NM_001271999, NM_001272001DONSON NM_017613 0.0754 DPEP2 Hs.372633 NM_022355 0.11 DPP7 NM_0133790.236 DPYD Hs.335034 NM_000110, NM_001160301 0.133 DPYSL2 Hs.593187NM_001197293, NM_001386, NM_001244604 0.106 DSN1 Hs.632268 NM_001145318,NM_001145315, NM_001145317, 0.0767 NM_001145316, NM_024918 DTL Hs.656473NM_016448, NM_001286229, NM_001286230 0.234 DUSP10 Hs.497822 NM_0072070.162 DUSP16 NM_030640 0.238 DUSP22 NM_020185, NM_001286555 0.0735 DUSP4NM_001394, NM_057158 0.00463 DVL2 Hs.118640 NM_004422 0.0847 DYNLL1Hs.5120 NM_001037494, NM_001037495, NM_003746 0.195 EAPP Hs.433269NM_018453 0.246 EBI3 NM_005755 0.00226 EBI3 0.00226 EED NM_003797,NM_152991, NM_001308007 0.0798 EEF1D NM_001195203, NM_032378,NM_001130056, 0.101 NM_001289950, NM_001960, NM_001130053, NM_001130055,NM_001130057 EFS Hs.24587 NM_005864, NM_032459, NM_001277174 0.0594EGFL6 Hs.12844 NM_015507, NM_001167890 0.229 EIF2C4 0.158 ELL2 Hs.192221NM_012081 0.0455 EMB Hs.561411 NM_198449 0.24 EMP1 NM_001423 0.0144ENPP2 Hs.190977 NM_006209, NM_001130863, NM_001040092 0.0275 EPAS1Hs.468410 NM_001430 0.00636 EPB41L4B Hs.591901 NM_019114, NM_0184240.172 EPHA4 Hs.371218 NM_001304536, NM_001304537, NM_004438 0.157 ERGIC3NM_015966, NM_198398 0.207 ERI2 Hs.248437 NM_080663, NM_001142725 0.0552ERP29 NM_006817, NM_001034025 0.0113 ESPL1 Hs.153479 NM_012291 0.236ESR1 Hs.208124 NM_001291230, NM_001122741, NM_001291241, 0.192NM_000125, NM_001122742, NM_001122740 ESRRG NM_001134285, NM_001243511,NM_001243518, 0.195 NM_001438, NM_001243514, NM_001243513, NM_206595,NM_001243506, NM_001243510, NM_001243507, NM_001243512, NM_001243509,NM_001243515, NM_001243519, NM_206594 ETV3 NM_001145312, NM_005240 0.208EVI5 Hs.594434 NM_005665, NM_001308248 0.0127 FADS1 Hs.503546, NM_0134020.16 Hs.739285 FADS2 NM_001281501, NM_001281502, NM_004265 0.0243 FAHNM_000137 0.131 FAIM2 Hs.567424 NM_012306 0.215 FAIM3 0.128 FAM125B0.0735 FAM134B Hs.481704 NM_019000, NM_001034850 0.0128 FAM134CHs.632262 NM_178126 0.107 FAM40B 0.00562 FAM46C Hs.356216 NM_0177090.241 FAM65B Hs.559459 NM_014722, NM_015864, NM_001286447, 0.0965NM_001286445, NM_001286446 FANCI Hs.513126 NM_018193, NM_001113378 0.133FAU NM_001997 0.137 FDPS NM_001135822, NM_001242824, NM_002004, 0.0852NM_001135821, NM_001242825 FGD3 Hs.411081 NM_001083536, NM_001286993,NM_033086 0.125 FGF21 Hs.283015 NM_019113 0.249 FHIT Hs.655995NM_002012, NM_001166243 0.0505 FKBP11 NM_001143781, NM_016594,NM_001143782 0.0128 FLI1 Hs.504281 NM_002017, NM_001167681,NM_001271012, 0.0965 NM_001271010 FLT1 Hs.594454 NM_002019,NM_001160030, NM_001159920, 0.0243 NM_001160031 FLT3LG NM_001204502,NM_001459, NM_001278637, 0.0124 NM_001278638, NM_001204503 FLVCR2Hs.509966 NM_017791, NM_001195283 0.149 FMNL2 Hs.654630 NM_052905 0.171FNBP1 NM_015033 0.142 FOXN3 NM_001085471, NM_005197 0.207 FOXP1NM_032682, NM_001244816, NM_001244815, 0.015 NM_001244814, NM_001244808,NM_001244812, NM_001012505, NM_001244813, NM_001244810 FXYD5 NM_014164,NM_144779, NM_001164605 0.0852 FXYD7 NM_022006 0.189 G0S2 NM_0157140.0594 GAB2 Hs.429434 NM_012296, NM_080491 0.0532 GAB3 Hs.496982NM_080612, NM_001282283, NM_001081573 0.136 GABARAPL1 Hs.524250NM_031412 0.162 GAD2 NM_001134366, NM_000818 0.129 GARS Hs.404321NM_002047 0.109 GATM Hs.75335 NM_001482 0.124 GBP5 Hs.513726 NM_052942,NM_001134486 0.168 GCET2 0.243 GEM Hs.654463 NM_181702, NM_0052610.00795 GK Hs.1466 NM_203391, NM_001128127, NM_000167, 0.0432NM_001205019 GLCCI1 Hs.131673 NM_138426 0.211 GLIPR2 Hs.493819NM_001287010, NM_001287013, NM_022343, 0.0466 NM_001287011,NM_001287014, NM_001287012 GMNN NM_001251990, NM_001251989, NM_015895,0.215 NM_001251991 GNG4 Hs.159711 NM_004485, NM_001098722, NM_0010987210.101 GNLY NM_006433, NM_001302758, NM_012483 0.184 GPC1 NM_002081 0.186GPD1L Hs.82432 NM_015141 0.099 GPKOW NM_015698 0.167 GPR114 0.17 GPR560.00463 GPRC5C Hs.446438 NM_022036, NM_018653 0.158 GPRIN3 NM_1982810.215 GPSM3 Hs.520046 NM_001276501, NM_022107 0.122 GRAMD3 Hs.363558,NM_001146319, NM_023927, NM_001146322, 0.0594 Hs.664026 NM_001146321,NM_001146320 GRAP NM_006613 0.158 GTF3C4 NM_012204 0.231 GTPBP1Hs.276925 NM_004286 0.0665 GYG1 Hs.477892, NM_004130, NM_001184720,NM_001184721 0.124 Hs.727448 GZMA NM_006144 0.0211 GZMH NM_001270780,NM_033423, NM_001270781 0.159 H19 Hs.533566 0.151 H1F0 Hs.745024NM_005318 0.0432 HAO2 Hs.659767 NM_001005783, NM_016527 0.17 HBS1LHs.378532 NM_006620, NM_001145158, NM_001145207 0.0528 HERPUD2 NM_0223730.236 HKDC1 Hs.522988 NM_025130 0.236 HLA-DPA1 Hs.347270 NM_033554,NM_001242524, NM_001242525 0.158 HLA-DQA2 NM_020056, NM_002122 0.179HLA-DQB2 Hs.731563 NM_001198858, NM_001300790 0.226 HLA-DRB5 NM_0021250.0645 HMGCR Hs.628096 NM_000859, NM_001130996 0.0466 HMGCS1NM_001098272, NM_002130 0.0666 HNRPLL 0.0566 HOXC8 NM_022658 0.142 HPGDHs.596913 NM_000860, NM_001145816, NM_001256301, 0.0276 NM_001256306,NM_001256307, NM_001256305 HPS5 Hs.437599 NM_007216, NM_181507,NM_181508 0.167 HSD11B1 NM_005525, NM_001206741, NM_181755 0.0502HSD17B11 Hs.594923 NM_016245 0.156 HSD17B12 Hs.132513 NM_016142 0.143HSPA1L Hs.690634 NM_005527 0.193 HSPD1 NM_002156, NM_199440 0.139 ICAM1Hs.643447 NM_000201 0.132 ICAM2 NM_000873, NM_001099789, NM_001099788,0.0847 NM_001099786, NM_001099787 ICAM3 Hs.654563 NM_002162 0.229 IER2Hs.501629 NM_004907 0.158 IER3 NM_003897 0.206 IFI44 NM_006417 0.223IFIH1 Hs.163173 NM_022168 0.0524 IGBP1 Hs.496267 NM_001551 0.151 IGSF3Hs.171057 NM_001007237, NM_001542 0.0466 IL17RA Hs.48353 NM_014339,NM_001289905 0.135 IL1A NM_000575 0.00562 IL1RAP Hs.478673 NM_001167929,NM_001167928, NM_002182, 0.0524 NM_134470, NM_001167930, NM_001167931IL1RAPL1 Hs.658912 NM_014271 0.167 IL1RN NM_173843, NM_173841,NM_000577, NM_173842 0.0168 IL21 NM_021803, NM_001207006 0.167 IL32NM_001012718, NM_004221, NM_001012633, 0.177 NM_001012631, NM_001012635,NM_001012634, NM_001012632, NM_001012636 IL8 0.234 INPP4B NM_003866,NM_001101669 0.0965 IRF4 NM_002460, NM_001195286 0.142 IRF6 Hs.591415NM_006147, NM_001206696 0.0699 IRF8 NM_002163 0.114 ISG20 Hs.459265NM_001303234, NM_001303233, NM_002201, 0.0354 NM_001303237 ITGA6Hs.133397 NM_000210, NM_001079818 0.0441 ITGAE Hs.513867 NM_002208 0.192ITPA NM_033453, NM_181493, NM_001267623 0.16 ITPK1 NM_001142594,NM_001142593, NM_014216 0.12 JUN Hs.696684 NM_002228 0.141 JUNB Hs.25292NM_002229 0.0455 KAZALD1 Hs.733496 NM_030929 0.214 KCNK1 NM_002245 0.104KCNK5 Hs.444448 NM_003740 0.246 KCNQ1 NM_000218, NM_181798 0.205 KIFC1Hs.436912 NM_002263 0.234 KIT Hs.479754 NM_001093772, NM_000222 0.0466KLF2 Hs.744182 NM_016270 0.166 KLF3 Hs.298658 NM_016531 0.24 KLF4Hs.376206 NM_004235 0.136 KLF7 Hs.59908 NM_003709, NM_001270943,NM_001270942, 0.203 NM_001270944 KLRB1 Hs.169824 NM_002258 0.00226 KLRC1Hs.512576 NM_001304448, NM_002259, NM_213657, 0.0455 NM_007328,NM_213658 KLRD1 Hs.562457, NM_002262, NM_007334, NM_001114396 0.158Hs.668357 KRT72 Hs.662013 NM_001146225, NM_001146226, NM_080747 0.212LAIR1 NM_001289026, NM_001289027, NM_002287, 0.238 NM_021706,NM_001289025, NM_001289023 LAMB3 Hs.497636 NM_000228, NM_001127641,NM_001017402 0.0642 LCLAT1 Hs.468048 NM_001304445, NM_001002257,NM_182551 0.234 LIF Hs.2250 NM_002309, NM_001257135 0.0837 LIMA1Hs.525419 NM_001243775, NM_001113546, NM_016357, 0.0774 NM_001113547LITAF Hs.459940 NM_001136473, NM_004862, NM_001136472 0.0747 LMCD1NM_014583, NM_001278235, NM_001278233, 0.0127 NM_001278234 LMNAHs.594444 NM_001282625, NM_005572, NM_170707, 0.0166 NM_001282626,NM_001257374, NM_001282624, NM_170708 LMNB2 Hs.538286 NM_032737 0.144LOC282997 0.198 LOC728392 0.12 LOC728855 0.0567 LRRC16A Hs.649550NM_001173977, NM_017640 0.0756 LSAMP Hs.26409 NM_002338 0.223 LTA Hs.36NM_001159740, NM_000595 0.135 LYAR Hs.425427 NM_017816, NM_0011457250.0882 MAP2K5 NM_145160, NM_002757, NM_001206804 0.107 MAP2K6 Hs.463978NM_002758 0.0594 MATK Hs.631845 NM_139354, NM_139355, NM_002378 0.177MBP Hs.551713 NM_001025081, NM_001025090, NM_001025092, 0.192 NM_002385,NM_001025100, NM_001025101 MCAM NM_006500 0.245 MCM10 Hs.198363NM_018518, NM_182751 0.232 MCM2 Hs.477481 NM_004526 0.119 MCM4NM_182746, NM_005914 0.144 MCTP2 Hs.33368 NM_001159643, NM_018349,NM_001159644 0.147 ME3 Hs.199743 NM_001014811, NM_001161586, NM_0066800.136 MEST NM_177524, NM_001253901, NM_001253902, 0.192 NM_001253900,NM_002402, NM_177525 METTL13 Hs.494705 NM_014955, NM_001007239,NM_015935 0.207 METTL7A Hs.744021 NM_014033 0.0605 MFNG Hs.517603NM_002405, NM_001166343 0.061 MGAT4C Hs.589093, NM_013244 0.0544Hs.739389 MICAL2 Hs.501928, NM_014632, NM_001282663, NM_001282665, 0.158Hs.735627 NM_001282666, NM_001282667, NM_001282664 MID1IP1 Hs.522605NM_001098790, NM_021242, NM_001098791 0.229 MIR155HG Hs.697120 0.0386MIS18A Hs.190518 NM_018944 0.0906 MLEC Hs.701392, NM_001303627,NM_014730, NM_001303628 0.244 Hs.744910 MLH1 Hs.195364 NM_000249,NM_001258271, NM_001258274, 0.159 NM_001167618, NM_001167617,NM_001167619, NM_001258273 MMP19 NM_002429, NM_001272101 0.165 MPP1Hs.496984 NM_001166460, NM_002436, NM_001166461, 0.212 NM_001166462 MRC2Hs.7835 NM_006039 0.215 MRPL39 NM_017446, NM_080794 0.151 MRPS17Hs.44298 NM_015969 0.0524 MT1G NM_005950, NM_001301267 0.214 MTHFD1Hs.652308 NM_005956 0.0524 MTHFD2 NM_006636 0.0924 MTMR4 Hs.514373NM_004687 0.171 MYB Hs.606320, NM_001130173, NM_001130172, NM_005375,0.0528 Hs.626299 NM_001161657, NM_001161656, NM_001161658, NM_001161659,NM_001161660 MYL6 NM_021019, NM_079423 0.137 MYO1C Hs.286226NM_001080779, NM_001080950, NM_033375 0.128 MYO1F Hs.465818 NM_0123350.0965 MYOF Hs.602086 NM_133337, NM_013451 0.0243 NAB2 Hs.159223NM_005967 0.0128 NCAPD2 Hs.5719 NM_014865 0.233 NCAPD3 NM_015261 0.236NCAPH NM_015341, NM_001281710, NM_001281711, 0.17 NM_001281712 NCKAP1Hs.603732 NM_013436, NM_205842 0.0111 NDFIP2 Hs.525093 NM_001161407,NM_019080 0.0528 NELL2 Hs.505326 NM_001145108, NM_006159, NM_001145110,0.0558 NM_001145107, NM_001145109 NFKBIZ Hs.319171 NM_001005474,NM_031419 0.181 NHSL2 Hs.397836, NM_001013627 0.192 Hs.660859 NINJ2NM_016533, NM_001294345, NM_001294346 0.061 NKG7 NM_005601 0.123 NKIRAS1Hs.173202 NM_020345 0.201 NMT2 NM_004808, NM_001308295 0.0842 NOGHs.248201 NM_005450 0.171 NOSIP Hs.7236 NM_001270960, NM_015953 3.65E−05NPC2 Hs.433222 NM_006432 0.156 NPY NM_000905 0.0466 NR1D2 Hs.37288NM_001145425, NM_005126 0.139 NR2E1 Hs.157688 NM_003269, NM_0012861020.0871 NR3C2 Hs.163924 NM_000901, NM_001166104 0.0645 NR4A3 Hs.279522NM_173199, NM_006981, NM_173200 0.171 NSMCE1 Hs.284295 NM_145080 0.0711NUP205 Hs.743250 NM_015135 0.162 OASL NM_003733, NM_198213, NM_0012618250.192 ODC1 NM_002539, NM_001287188, NM_001287190, 0.139 NM_001287189OLFM2 NM_058164, NM_001304348, NM_001304347 0.229 ORC6 NM_014321 0.0798OSBPL8 Hs.430849 NM_020841, NM_001003712 0.227 OTUD7B NM_020205 0.0487P2RX4 NM_002560, NM_001256796, NM_001261397, 0.0671 NM_001261398 P4HA2Hs.519568 NM_001142598, NM_001142599, NM_001017974, 0.107 NM_001017973,NM_004199 PACSIN3 Hs.334639 NM_016223, NM_001184974, NM_001184975 0.226PAICS Hs.518774 NM_001079525, NM_001079524, NM_006452 0.167 PAMHs.369430, NM_001177306, NM_138822, NM_138821, 0.218 Hs.738567NM_000919, NM_138766 PANX2 Hs.440092 NM_001160300, NM_052839 0.0128PARP8 Hs.369581 NM_001178055, NM_024615, NM_001178056 0.122 PBX4Hs.466257 NM_025245 0.211 PCSK5 Hs.368542 NM_006200, NM_001190482 0.0473PDCD1 Hs.158297 NM_005018 0.0747 PDE10A Hs.348762, NM_006661,NM_001130690 0.215 Hs.638546 PDGFRB Hs.509067 NM_002609 0.0628 PECAM1Hs.376675 NM_000442 0.0666 PELP1 Hs.744899 NM_014389, NM_001278241 0.192PER1 Hs.445534 NM_002616 0.0674 PEX16 NM_057174, NM_004813 0.0666 PFDN5NM_002624, NM_145897 0.135 PFKM Hs.75160 NM_001166686, NM_000289,NM_001166688, 0.0961 NM_001166687 PGAP1 Hs.229988 NM_024989 0.158 PGCP0.186 PHEX NM_001282754, NM_000444 0.149 PHF6 Hs.356501 NM_032458,NM_001015877, NM_032335 0.202 PHLPP1 Hs.465337 NM_194449 0.0853 PIK3C2GHs.22500 NM_001288772, NM_001288774, NM_004570 0.0629 PINK1 Hs.389171NM_032409 0.24 PION 0.187 PITPNC1 Hs.591185 NM_181671, NM_012417 0.00827PKMYT1 Hs.732385, NM_182687, NM_004203, NM_001258451, 0.246 Hs.734466NM_001258450 PLAC8 Hs.546392 NM_016619, NM_001130716, NM_0011307150.0653 PLAGL2 Hs.154104 NM_002657 0.174 PLCG2 NM_002661 0.118 PLCL1NM_006226 0.107 PLCL2 Hs.202010, NM_001144382, NM_015184 0.141 Hs.741267PLIN2 NM_001122 0.0747 PLK2 Hs.398157 NM_006622, NM_001252226 0.205PLXNB2 Hs.3989 NM_012401 0.0064 PLXND1 Hs.301685 NM_015103 0.229 PMAIP1Hs.96 NM_021127 0.0316 PMCH NM_002674 0.0126 PNLIPRP1 Hs.73923NM_006229, NM_001303135 0.137 PNMA1 Hs.194709 NM_006029 0.11 POU2AF1Hs.654525, NM_006235 0.0127 Hs.733573 POU6F1 Hs.555886 NM_002702 0.229PPCDC Hs.458922, NM_021823, NM_001301103, NM_001301101, 0.00748Hs.640486 NM_001301102, NM_001301104, NM_001301105 PPFIBP2 Hs.655714,NM_003621, NM_001256568, NM_001256569 0.0544 Hs.739217 PPP1R15AHs.631593 NM_014330 0.0645 PPP2R2B Hs.739387 NM_181678, NM_181674,NM_181675, NM_181676, 0.061 NM_001271899, NM_181677, NM_001271900,NM_001271948 PPP2R3C Hs.530712 NM_017917, NM_001305156, NM_0013051550.167 PPP2R5C Hs.368264, NM_001161726, NM_178586, NM_178587, 0.158Hs.679341 NM_002719, NM_001161725 PPP3CA Hs.435512 NM_000944,NM_001130692, NM_001130691 0.0679 PQBP1 NM_001167990, NM_005710,NM_001032384, 0.141 NM_144495, NM_001032381, NM_001167989, NM_001032382,NM_001032383 PRC1 Hs.366401 NM_003981, NM_199413, NM_001267580 0.0642PRDM1 Hs.436023 NM_001198, NM_182907 0.0127 PRF1 Hs.2200 NM_001083116,NM_005041 0.192 PRKAR1B Hs.520851 NM_001164761, NM_001164758, NM_002735,0.167 NM_001164760, NM_001164759, NM_001164762 PRKCDBP NM_145040 0.0181PRKCH Hs.333907, NM_006255 0.0889 Hs.630857 PRKCQ Hs.498570 NM_006257,NM_001282644, NM_001242413, 0.115 NM_001282645 PRKD3 Hs.660757 NM_0058130.195 PRKG2 Hs.570833 NM_001282485, NM_006259, NM_001282483, 0.205NM_001282482, NM_001282481, NM_001282480 PRNP Hs.472010 NM_001080121,NM_001271561, NM_001080122, 0.236 NM_000311, NM_183079, NM_001080123PRR5 NM_015366, NM_001198721, NM_181333, 0.0135 NM_001017528,NM_001017530, NM_001017529 PRSS23 Hs.25338, NM_001293180, NM_001293178,NM_007173, 0.158 Hs.729257 NM_001293179 PSMA5 Hs.485246 NM_002790,NM_001199773, NM_001199774, 0.213 NM_001199772 PSMB9 NM_002800 0.234PSMC2 Hs.437366 NM_001204453, NM_002803 0.246 PSMD11 NM_001270482,NM_002815 0.206 PSMD14 Hs.740477 NM_005805 0.131 PTGFRN Hs.418093NM_020440 0.215 PTPN12 Hs.61812 NM_002835, NM_001131008, NM_0011310090.205 PTPN14 Hs.193557, NM_005401 0.124 Hs.688910 PTPN3 Hs.436429,NM_001145368, NM_002829, NM_001145369, 0.135 Hs.698275 NM_001145370PTPN4 Hs.469809 NM_002830 0.187 PTPN6 Hs.63489 NM_080548, NM_002831,NM_080549 0.0174 PTTG1 NM_004219, NM_001282383, NM_001282382 0.235 PUS7Hs.520619 NM_019042 0.127 PVR NM_006505, NM_001135769, NM_001135768,0.0064 NM_001135770 PYCARD NM_013258, NM_145182 0.0843 PYCR1 NM_006907,NM_153824, NM_001282279, 0.235 NM_001282281, NM_001282280 RAB37NM_175738, NM_001163989, NM_001006638, 0.192 NM_001163990 RACGAP1Hs.505469 NM_013277, NM_001126104, NM_001126103 0.167 RAD51NM_001164270, NM_002875, NM_133487, 0.246 NM_001164269 RAP1GAP2Hs.499659, NM_015085, NM_001100398 0.167 Hs.685132 RARRES3 NM_0045850.116 RASAL1 Hs.528693 NM_001193520, NM_004658, NM_001193521, 0.125NM_001301202 RASGEF1A Hs.125293 NM_001282862, NM_145313 0.0831 RASGRP2Hs.99491 NM_001098671, NM_001098670, NM_153819 0.115 RASGRP4 Hs.130434NM_001146202, NM_001146204, NM_001146205, 0.129 NM_170604, NM_001146207,NM_001146203, NM_001146206 RBMS1 Hs.470412, NM_016836, NM_002897 0.0466Hs.654231 REEP2 NM_001271803, NM_016606 0.135 REPIN1 Hs.647086NM_001099695, NM_014374, NM_013400, 0.243 NM_001099696 RGS1 Hs.75256NM_002922 0.149 RGS12 NM_198229, NM_002926, NM_198227 0.192 RGS9Hs.664380 NM_001165933, NM_001081955, NM_003835 0.222 RHOB Hs.502876NM_004040 0.0842 RNF125 Hs.633703 NM_017831 0.135 RNF19A Hs.292882,NM_015435, NM_183419, NM_001280539 0.0544 Hs.735657 RPLP2 NM_0010040.111 RPS20 NM_001146227, NM_001023 0.189 RPS27 NM_001030 0.0505 RPS28NM_001031 0.0135 RPS6KA3 Hs.445387 NM_004586 0.189 RSAD2 Hs.17518NM_080657 0.229 RTF1 Hs.511096 NM_015138 0.219 RYBP NM_012234 0.156S100A4 NM_019554, NM_002961 0.00133 S1PR1 Hs.154210 NM_001400 0.151S1PR4 Hs.662006, NM_003775 0.0505 Hs.688059 SALL2 Hs.416358, NM_005407,NM_001291446, NM_001291447 0.0685 Hs.745364 SCD Hs.558396 NM_0050630.0265 SCGB1A1 NM_003357 0.152 SCML4 NM_001286408, NM_001286409,NM_198081 0.0243 SDC4 Hs.632267 NM_002999 0.156 SDK2 Hs.435719NM_001144952 0.0307 SECTM1 Hs.558009 NM_003004 0.0735 SELPLG NM_003006,NM_001206609 0.245 SEMA3B Hs.82222 NM_001290060, NM_001005914,NM_001290061, 0.0477 NM_001290063, NM_001290062, NM_004636 SEPT3Hs.120483 NM_145733, NM_019106 0.0175 SERPINF1 NM_002615 0.11 SERPINF2Hs.159509 NM_001165921, NM_001165920, NM_000934 0.21 SFMBT2 Hs.407983NM_001018039, NM_001029880 0.0871 SFXN1 NM_022754 0.174 SGCB Hs.438953NM_000232 0.12 SGTB Hs.482301 NM_019072 0.246 SH2B2 Hs.489448 NM_0209790.0127 SHF Hs.310399 NM_001301169, NM_001301168, NM_138356, 0.0567NM_001301170, NM_001301171 SIGIRR Hs.501624 NM_001135054, NM_021805,NM_001135053 0.143 SIGLEC9 Hs.245828 NM_014441, NM_001198558 0.246SIPA1L1 Hs.654657 NM_001284245, NM_015556, NM_001284247, 0.215NM_001284246 SKAP1 Hs.316931 NM_003726, NM_001075099 0.0127 SLA2Hs.713578 NM_032214, NM_175077 0.227 SLAMF1 NM_003037 0.246 SLC11A1Hs.591607 NM_000578 0.144 SLC14A1 NM_001308278, NM_015865, NM_001146036,0.243 NM_001128588, NM_001308279, NM_001146037 SLC16A1 Hs.75231NM_003051, NM_001166496 0.18 SLC1A4 Hs.654352 NM_001193493, NM_0030380.246 SLC1A5 Hs.631582 NM_005628, NM_001145145, NM_001145144 0.218SLC22A17 Hs.373498 NM_001289050, NM_016609, NM_020372 0.0366 SLC25A20Hs.13845 NM_000387 0.22 SLC27A2 Hs.11729 NM_003645, NM_001159629 0.0274SLC29A1 Hs.25450 NM_001304463, NM_001078175, NM_001078177, 0.00562NM_001304465, NM_001304466, NM_001304462 SLC2A1 Hs.473721 NM_0065160.0507 SLC2A3 Hs.419240 NM_006931 0.192 SLC35F2 Hs.524014 NM_0175150.0567 SLC39A1 Hs.7854 NM_001271958, NM_001271957, NM_014437, 0.144NM_001271959, NM_001271960, NM_001271961 SLC39A14 Hs.491232 NM_015359,NM_001135154, NM_001128431, 0.156 NM_001135153 SLC43A1 Hs.591952NM_003627, NM_001198810 0.192 SLC43A3 Hs.99962 NM_014096, NM_001278201,NM_199329, 0.015 NM_017611, NM_001278206 SLC46A3 NM_181785, NM_0011359190.0767 SLCO3A1 Hs.311187 NM_013272, NM_001145044 0.151 SMAP2 Hs.15200NM_022733, NM_001198978, NM_001198980, 0.164 NM_001198979 SMTNNM_134270, NM_006932, NM_134269, 0.162 NM_001207018, NM_001207017 SMYD5Hs.631882 NM_006062 0.246 SNAI2 Hs.360174 NM_003068 0.0502 SNTB1Hs.46701 NM_021021 0.162 SNTG1 NM_001287813, NM_018967, NM_0012878140.0585 SORD NM_003104 0.0455 SPATA7 NM_018418, NM_001040428 0.0965SPATS2L Hs.120323, NM_001282735, NM_015535, NM_001100422, 0.061Hs.734045 NM_001282743, NM_001100424, NM_001100423, NM_001282744 SPINK2NM_021114, NM_001271718, NM_001271720, 0.0642 NM_001271722, NM_001271721SPINT1 Hs.233950 NM_003710, NM_001032367, NM_181642 0.246 SPSB3Hs.592080 NM_080861 0.122 SQLE Hs.71465 NM_003129 0.0502 SREK1IP1Hs.69504 NM_173829 0.163 SSBP3 Hs.733025 NM_145716, NM_001009955,NM_018070 0.0112 SSH1 NM_018984, NM_001161330, NM_001161331 0.163 SSR2NM_003145 0.0544 ST6GALNAC2 Hs.592105 NM_006456 0.224 ST8SIA1 Hs.408614NM_003034 0.0699 STAMBP Hs.469018, NM_213622, NM_006463, NM_201647 0.11Hs.732857 STAP1 Hs.435579 NM_012108 0.0685 STAT6 Hs.524518 NM_003153,NM_001178078, NM_001178081, 0.158 NM_001178079, NM_001178080 STILHs.525198, NM_001282936, NM_003035, NM_001048166, 0.205 Hs.673209NM_001282937, NM_001282939, NM_001282938 STIP1 Hs.337295, NM_001282652,NM_006819, NM_001282653 0.223 Hs.618350 STK38 Hs.409578 NM_001305102,NM_007271 0.0924 STMN3 NM_015894, NM_001276310 0.0735 STOM NM_004099,NM_001270526, NM_198194, 0.112 NM_001270527 STX1A Hs.647024 NM_004603,NM_001165903 0.222 STX6 Hs.518417 NM_005819, NM_001286210 0.236 SV2AHs.516153 NM_014849 0.216 SVIL Hs.499209 NM_003174, NM_021738 0.243 SYT1Hs.310545 NM_001135805, NM_005639, NM_001291901, 0.0645 NM_001135806SYTL1 NM_032872, NM_001193308 0.23 SYTL2 Hs.369520 NM_206929, NM_206930,NM_001162951, 0.0502 NM_001162952, NM_001289610, NM_001289608,NM_032943, NM_001162953 SYTL3 NM_001242395, NM_001009991, NM_001242384,0.129 NM_001242394 TAF7 Hs.438838 NM_005642 0.0836 TARP 0.22 TARSNM_001258437, NM_152295, NM_001258438 0.158 TBCC NM_003192 0.246 TBX15Hs.146196 NM_152380 0.00865 TCEAL4 NM_001300901, NM_024863,NM_001006935, 0.0594 NM_001006937 TCF7 NM_003202, NM_201634,NM_001134851, 0.114 NM_213648, NM_201632 TERT Hs.492203 NM_198253,NM_001193376 0.0311 TGFB1 Hs.645227 NM_000660 0.12 TGFBR3 NM_001195684,NM_001195683, NM_003243 0.188 TIGIT Hs.421750 NM_173799 0.234 TJP3Hs.25527 NM_001267560, NM_001267561 0.248 TLE4 NM_007005, NM_001282760,NM_001282748, 0.0302 NM_001282749, NM_001282753 TMEM194A 0.106 TMEM212Hs.642307 NM_001164436 0.102 TMEM48 0.171 TMEM5 Hs.216386 NM_014254,NM_001278237 0.178 TMEM71 Hs.293842 NM_144649, NM_001145153 0.234 TMEM80NM_001042463, NM_174940, NM_001276274, 0.246 NM_001276253 TMEM9BNM_001286094, NM_020644, NM_001286095 0.192 TMPRSS6 Hs.370885 NM_153609,NM_001289001, NM_001289000 0.0585 TNFAIP1 Hs.76090 NM_021137 0.167TNFRSF11A NM_001270951, NM_003839, NM_001278268, 0.0605 NM_001270949,NM_001270950 TNFRSF18 Hs.212680 NM_148901, NM_004195, NM_148902 0.0366TNFRSF25 Hs.462529 NM_148967, NM_148970, NM_148966, 0.163 NM_003790,NM_148965 TNFRSF4 Hs.129780 NM_003327 0.0337 TNFRSF8 Hs.1314 NM_001243,NM_001281430 0.106 TNFRSF9 Hs.86447 NM_001561 0.0524 TOX2 Hs.26608NM_001098797, NM_001098796, NM_032883, 0.0107 NM_001098798 TP53INP2Hs.516994 NM_021202 0.139 TPCN1 Hs.524763 NM_017901, NM_001143819,NM_001301214 0.181 TPK1 Hs.660232 NM_022445, NM_001042482 0.24 TPMTHs.444319 NM_000367 0.232 TRAPPC6A NM_024108, NM_001270893,NM_001270891, 0.0638 NM_001270892 TRIB1 Hs.444947 NM_025195,NM_001282985 0.074 TRIB2 Hs.467751 NM_021643 0.0181 TRIM25 Hs.528952NM_005082 0.143 TRIP10 Hs.515094 NM_004240, NM_001288962, NM_0012889630.149 TRPM3 NM_001007471, NM_020952, NM_206946, 0.195 NM_206945,NM_001007470, NM_206948, NM_024971, NM_206944, NM_206947 TSPAN18Hs.385634 NM_130783 0.129 TSPAN32 NM_139022 0.0502 TTC21B Hs.310672NM_024753 0.243 TTC39C Hs.733420 NM_153211, NM_001243425, NM_001135993,0.00494 NM_001292030 TTLL4 Hs.471405 NM_014640 0.158 TUBB Hs.636480NM_001293213, NM_178014, NM_001293215, 0.236 NM_001293216, NM_001293212TUBB2B Hs.300701 NM_178012 0.0386 TUBB6 Hs.193491, NM_001303524,NM_032525, NM_001303529, 0.00952 Hs.744066 NM_001303526, NM_001303525UBA52 Hs.5308 NM_003333, NM_001033930 0.0902 UBASH3B Hs.444075 NM_0328730.0666 UBL3 Hs.145575 NM_007106 0.0929 UHRF1BP1L Hs.620701 NM_015054,NM_001006947 0.0666 UNC119 Hs.410455 NM_005148, NM_054035 0.168 URGCPHs.663312 NM_017920, NM_001077664, NM_001290075, 0.141 NM_001290076,NM_001077663 USP22 Hs.462492 NM_015276 0.0745 USP25 NM_013396,NM_001283042, NM_001283041 0.232 USP51 Hs.40061 NM_201286 0.186 UXS1Hs.730756 NM_001253875, NM_025076, NM_001253876 0.167 UXT NM_004182,NM_153477 0.0826 VDAC3 NM_001135694, NM_005662 0.158 VDR Hs.524368NM_001017535, NM_001017536, NM_000376 0.124 VIPR1 Hs.348500,NM_001251882, NM_001251885, NM_004624, 0.0642 Hs.683175 NM_001251883,NM_001251884 VNN2 Hs.293130, NM_004665, NM_078488, NM_001242350 0.129Hs.740120 VSIG1 Hs.177164 NM_182607, NM_001170553 0.0709 WASF2 Hs.469244NM_006990, NM_001201404 0.137 WEE1 NM_003390, NM_001143976 0.0594 WIPI1Hs.463964 NM_017983 0.171 WNT10A Hs.121540 NM_025216 0.0502 XYLT1Hs.22907 NM_022166 0.0278 YPEL1 NM_013313 0.195 YWHAG Hs.744840NM_012479 0.0783 ZBP1 Hs.302123 NM_001160417, NM_030776, NM_001160418,0.218 NM_001160419 ZBTB20 NM_001164343, NM_001164347, 0.00562NM_001164345, NM_001164342, NM_015642, NM_001164344, NM_001164346 ZBTB32Hs.99430, NM_014383 0.0128 Hs.736841 ZC3H12A Hs.656294 NM_025079 0.198ZC3H12C Hs.376289 NM_033390 0.0628 ZC3H12D NM_207360 0.0709 ZEB2Hs.34871 NM_014795, NM_001171653 0.0594 ZFP161 0.0965 ZFP36L2 Hs.503093NM_006887 0.218 ZHX2 Hs.377090 NM_014943 0.144 ZNF267 NM_003414 0.127ZNF282 Hs.729056 NM_003575, NM_001303481 0.121 ZNF506 NM_001099269,NM_001145404 0.195 ZNF587 Hs.744891 NM_032828, NM_001204817 0.236 ZNF652NM_014897, NM_001145365 0.0429 ZNF688 NM_145271, NM_001024683 0.229ZNF704 Hs.434957, NM_001033723 0.215 Hs.730558 ZNRF1 Hs.427284 NM_0322680.164

Example 3: Prognostic Flow Cytometry-Based Assays

Prognostic flow cytometry-based assays are developed to screen subjectswith cancer (e.g., patients with a hematological cancer such as ALL andCLL) for CAR-expressing cell (e.g., T cell, NK cell) therapy, e.g., CD19CAR-expressing cell therapy as described herein such as, e.g., CTL019therapy. In some embodiments, subjects are participating in clinicaltrials.

A sample (e.g., a blood sample) is isolated from a patient and afluorescent flow cytometry-based assay is performed screening for one ormore cell surface or secreted biomarkers described in Examples 1 and 2.An exemplary list of markers that are measured, e.g., by flow cytometryif cell surface-expressed, or by ELISA if secreted, and whose expressionvalues predict patient response to CAR-expressing cell (e.g., T cell, NKcell) therapy, e.g., CD19 CAR-expressing cell therapy as describedherein such as, e.g., CTL019 therapy includes, but is not limited to,genes listed in Table 8.

TABLE 8 Exemplary markers measured that predict patient response toCAR-expressing cell therapy Gene Unigene Accession No. ATP1B3 Hs.477789NM_001679 CCL17 Hs.546294 NM_002987 CCL3 Hs.514107 NM_002983 CCL4Hs.75703 NM_002984 CCR1 Hs.301921 NM_001295 CD40LG Hs.592244 NM_000074CD58 Hs.34341 NM_001144822, NM_001779 CD70 Hs.715224, Hs.501497NM_001252 CD80 Hs.838 NM_005191 CSF1 Hs.591402 NM_000757, NM_172212,NM_172211, NM_172210 FCER2 Hs.465778 NM_002002 GPR56 Hs.513633NM_001145773, NM_001145774, NM_001145771, NM_001145772, NM_005682,NM_201525, NM_001145770 HAVCR1 Hs.129711 NM_001099414, NM_012206 HLA-DMAHs.351279 NM_006120 HLA-DPA1 Hs.347270 NM_033554 HLA-DRA Hs.520048NM_019111 HLA-DRB1 Hs.716081, Hs.696211, NM_002124, NM_021983,XM_002346251 Hs.723344, Hs.534322 HLA-DRB5 Hs.534322 NM_002125 ICAM3Hs.654563 NM_002162 IFNAR2 Hs.708195 NM_207584, NM_207585, NM_000874IFNG Hs.856 NM_207585 IGF1R Hs.643120, Hs.714012 NM_000875 IL10Hs.193717 NM_000572 IL13 Hs.845 NM_002188 IL15RA Hs.524117 NM_002189,NM_172200 IL21 Hs.567559 NM_021803 IL2RA Hs.231367 NM_000417 IL2RBHs.474787 NM_000878 IL3 Hs.694 NM_000588 IL4 Hs.73917 NM_000589,NM_172348 IL5 Hs.2247 NM_000879 IL6ST Hs.532082 NM_002184, NM_175767 IL9Hs.960 NM_000590 ITGA6 Hs.133397 NM_000210, NM_001079818 KIT Hs.479754NM_000222, NM_001093772 LAIR1 Hs.572535 NM_001289023, NM_001289025,NM_001289026, NM_001289027, NM_002287, NM_021706 NFATC1 Hs.534074,Hs.701518 NM_001278669, NM_001278670, NM_001278672, NM_001278673,NM_001278675, NM_006162, NM_172387, NM_172388, NM_172389, NM_172390 SELLHs.728756 NM_000655 SELP Hs.73800 NM_003005 SIRPG Hs.590883NM_001039508, NM_018556, NM_080816 STAT6 Hs.524518 NM_001178078,NM_001178079, NM_001178080, NM_001178081, NM_003153 TFRC Hs.529618NM_001128148, NM_003234 TIMD4 Hs.334907 NM_001146726, NM_138379 TNFRSF1BHs.256278 NM_001066 TNFRSF9 Hs.86447, Hs.738942 NM_001561

Example 4: Classifiers to Predict Class Membership

Based on the biological understanding, combinations of genes fromunbiased feature selection, gene sets, and selected genes of interestare used to further differentiate complete responders from partialresponders and non-responders. In an embodiment, combinations of genesfrom unbiased feature selection, gene sets, and selected genes ofinterest are used to further differentiate relapsers from non-relapsers.In an embodiment, classifiers are built based on all genes to predictclass membership. In an embodiment, predictions of class membershipfurther differentiate NR's, PR's, and CR's. In an embodiment,predictions of class membership further differentiate relapsers fromnon-relapsers. Alternatively or additionally, a classifier is builtwhich uses a subset of predetermined significant features. Significantfeatures include, but are not limited to, enriched meta-gene, a subsetof significantly differentially expressed genes in the meta-genes, andcombinations thereof.

Example 5: Cytokine Expression Signatures Predictive of CAR-ExpressingCell Potency

The present example describes the identification of exemplary cytokineexpression signatures that predict patient response to CAR-expressingcell (e.g., T cell, NK cell) therapy (e.g., a CD19 CAR-expressing cell(e.g., T cell, NK cell) therapy, e.g., a CTL019 therapy) in ChronicLymphoid Leukemia (CLL) and Acute Lymphoblastic Leukemia (ALL), for usein accordance with the present invention.

Among other things, the present Example describes novel cytokineexpression signatures that predict the potency of manufacturedCAR-expressing cell (e.g., T cell, NK cell) cell products based onsecreted cytokine profiles following activation in vitro.

In an embodiment, novel cytokine expression signatures described hereinpredict the potency of manufactured CAR-expressing cell (e.g., T cell,NK cell) products to kill target tumor cells.

In an embodiment, novel cytokine expression signatures described hereinare correlated with patient response to CAR-expressing cell (e.g., Tcell, NK cell) therapy (e.g. CD19 CAR-expressing cell (e.g., T cell, NKcell) therapy, e.g., a CTL019 CAR-expressing cell therapy) in CLL toimprove the CAR-expressing cell product prior to infusion in patients.

In an embodiment, novel cytokine expression signatures described hereinare used to assess manufactured CAR-expressing cell (e.g., T cell, NKcell) products (e.g., CD19 CAR-expressing cell products, e.g., CTL019product). In an embodiment, novel cytokine expression signaturesdescribed herein provide an endpoint in manufacturing processoptimiziation.

Novel cytokine expression signatures based on cytokine proteinexpression levels in manufactured CD19 CAR-expressing cell (e.g., Tcell, NK cell) product samples prior to re-infusion have been identifiedthat predict patient response to CD19 CAR-expressing cell (e.g., T cell,NK cell) therapy in Chronic Lymphoid Leukemia (CLL). The identifiedsignatures were discovered in a cytokine protein expression study ofmanufactured product samples prepared from 21 CLL subject samples. CLLsubject samples (21 total) were stratified as follows: CTL019manufactured product was derived from 6 patients that were completeresponders (CRs) to CTL019 therapy, 5 patients that were partialresponders (PRs), and 10 non-responders (NRs). Several cytokineexpression signatures discriminating responders from non-responders inmanfactured product were discovered using a Luminex® panel of 13cytokines.

The potency of CTL019 manufactured products from 21 CLL patients wereassessed in a tumor cell killing assay. Briefly, manufactured CTL019products were “activated” in vitro by CD19-expressing K562 (K562-19cells). Without wishing to be bound by a particular theory, CD19expressing K562 cells (e.g., K562-19 cells) mimic leukemic CD19expressing B-cells in CLL patients. CTL019 cells are engineered toidentify and kill cells that express CD19 antigen on their cell surfaceand CTL019-mediated killing of K562-19 cells serves as a proxy forasessing potency of CTL019-mediated killing of tumor cells.

Following CTL019 product activation, cytokine protein expressionprofiles were measured in the co-cultured media using a Luminex® panelof cytokines. The expression profiles of exemplary cytokines weremeasured and the potency of CTL019 cell products was correlated with theexpression of different cytokines. Exemplary cytokines considered inthis analysis are provided in Table 14.

TABLE 14 Exemplary cytokines Cytokine Entrez ID Official Gene SymbolCCL-20/MIP-3a 6364 CCL20 GM-CSF 1437 CSF2 IFNγ 3458 IFNG IL-10 3586 IL10IL-13 3596 IL13 IL-17a 3605 IL17A IL-2 3558 IL2 IL-21 59067 IL21 IL-43565 IL4 IL-5 3567 IL5 IL-6 3569 IL6 IL-9 3578 IL9 TNFα 7124 TNF

Novel cytokine expression profiles were then discovered using variousdata analytical approaches including 1) bi-clustering analysis; and 2)univariate analysis.

Cytokine expression data derived from the Luminex® assay werelog-normalized and subjected to bi-clustering analyses (hierarchicalclustering was performed using the complete linkage method).Bi-clustering analyses of cytokine expression in stimulated CTL019products and CLL patients yield four major clusters (a cut-off distance≤1.0, resulted in 4 clusters as shown in FIG. 17) and distinct subgroupsof CRs/PRs and NRs were identified. An exemplary heatmap ofbi-clustering of cytokine expression in stimulated CTL019 products andCLL patients is shown in FIG. 17. Surprisingly, two clusters (Cluster 1and Cluster 3) were almost exclusively comprised of CRs and PRs, whereasthe other two clusters (Cluster 2 and Cluster 4) contained predominantlyNRs. On average, cytokine expression levels were higher in CRs/PRsversus NRs (FIG. 17).

Next, a 3-group univariate analysis using ANOVA (analysis of variance)was performed that compared CRs versus PRs versus NRs. Statisticalsignificance was determined using a p-value cut-off of 0.05. Statisticalsignificance (e.g., p-values) of different cytokines to distinguish CRs,PRs and NRs are listed in Table 15.

TABLE 15 Statistical significance of different cytokines to distinguishCRs, PRs and NRs in a 3-group univariate analysis using ANOVA Cytokinep-value CCL20/MIP3a 0.001838 IL-17a 0.001857 IL-6 0.006017 TNFα 0.013499IL-2 0.034397 IL-21 0.055684 IL-5 0.075396 IL-10 0.08935 IL-9 0.098761IFNγ 0.137263 GM-CSF 0.191839 IL-4 0.197774 IL-13 0.222134

The 3-group model of univariate analysis identified 5 cytokines, e.g.,IL-17a, CCL-20/MIP3a, IL-6, IL-2, and TNFα, as statistically significantmarkers of response to CTL019 therapy in CLL patients (FIG. 18 and Table15). Exemplary results of log-normalized expressions of statisticallysignificant cytokines that distinguish CRs, PRs and NRs in CLL patientsare shown in FIG. 18.

Manufactured CTL019 product was evaluted by flow cytometry to determinepercentages of CAR+ cells. The 5 cytokines identified in the 3-groupmodel of univariate analysis were further correlated to the percentageof CAR+ cells in each of the manufactured CTL019 products. Exemplarycorrelation coefficients and corresponding p-values of cytokineexpression (derived from Luminex® panel discussed above) and percentagesof CAR+ cells (determined by flow cytometery) are provided in Table 16.

TABLE 16 Correlation coefficients and corresponding p-values of cytokineexpression and percentage of CAR+ cells Cytokine Correlation coefficientp. value IL-17a 0.278349 0.221794 IL-10 0.390318 0.08024 CCL20/MIP3a0.395273 0.076147 IL-5 0.494758 0.022598 IL-4 0.525982 0.014321 TNFα0.539276 0.011642 GM-CSF 0.588262 0.005032 IL-6 0.631841 0.002122 IFNγ0.660738 0.001112 IL-2 0.661608 0.001089 IL-21 0.674378 0.0008 IL-90.70858 0.000324 IL-13 7.53E−01 8.19E−05

The percentage of CAR+ cells in CTL019 product represents transductionefficiency. In CLL, the percent of CAR+ cells at pre-harvest levelsdistinguish responders (e.g., complete responders and partialresponders) from non-responders (NR). FIG. 21 depicts an exemplaryscatter plot showing percent of CAR+ cells (i.e., transduction rate) atpre-harvest for complete responders (CR) in red, partial responders (PR)in blue and non-responders (NR) in red. Transduction efficiencies weremeasured pre-harvest and correlated with subject response (e.g., CR, PR,or NR). The solid line represents a 15% transduction efficiency thatseparates the majority of non-responders from responders. Withoutwishing to be bound to a particular theory, these data indicate thatpre-harvest CAR transduction efficiency is a marker of response toCAR-expressing cell (e.g., T cell, NK cell) therapy in CLL.

Correlation analysis of IL17a with CCL20 cytokine expression wasperformed, and their association with clinical response was evaluated byscatter plot analyses. FIG. 19A depicts an exemplary scatter plotshowing log-normalized correlation of IL17A (y-axis) and CCL20 (x-axis)expression with a correlation coefficient of 0.928 and correspondingp-value of 1.36e-09. Dashed lines represent the classification boundaryfor separating NRs from CRs/PRs. Each dot in FIG. 19A represents a CLLpatient, and the cross-hatch (NR), black (PR) and white (CR) representthe clinical response. Classification boundary in FIG. 19A demonstratesthat the combination of IL-17a and CCL20 separates almost all NRs fromCRs/PRs, and PRs in turn are clustered separately from CRs. Among otherthings, these data demonstrate CAR+ cell expression of one or morecytokines listed in Table 16 predict clinical response.

Surprisingly, IL-17a and CCL-20 expression levels were not correlatedwith the percentage of CAR+ cells in the CTL019 product (representingtransduction efficiency). Without wishing to be bound by a particulartheory, these data indicate IL-17a and CCL-20 cytokine expression levelsare informative (e.g., predictive of response) with regard to potency ofa manufactured CAR-expressing cell (e.g., T cell, NK cell) product,e.g., a manufactured CD19 CAR-expressing cell product, in several ways.First, cytokine signatures are correlated with patient response toCTL019 CAR-expressing cell therapy in CLL. Therefore, cytokinesignatures described herein can be used to improve and/or modifyCAR-expressing cell (e.g., T cell, NK cell) product (e.g., a CD19CAR-expressing cell product such as, e.g., CTL019) prior to infusion inpatients, for greater clinical efficacy. Second, cytokine signaturesdescribed herein can be used to assess manufactured CAR-expressing cell(e.g., T cell, NK cell) products thereby providing, among other things,an end point in manufacturing process optimization.

In an embodiment, cytokine signatures described herein define thepotency of a CAR-expressing cell (e.g., T cell, NK cell) product. In anembodiment, cytokine signatures described herein are markers of responseto a CAR-expressing cell (e.g., T cell, NK cell) product in ahematological cancer (e.g., CLL or ALL).

In an embodiment, cytokine signatures described herein predict subjectresponse to a CAR-expressing cell (e.g., T cell, NK cell) product.

In an embodiment, cytokine signatures described in Table 16 predictsubject response to a CAR-expressing cell (e.g., T cell, NK cell)product.

In an embodiment, IL-17a and CCL-20 expression levels predict subjectresponse to a CAR-expressing cell (e.g., T cell, NK cell) product.

Example 6: Identification of Factors that Predict Subject Relapse toCD19 CAR-Expressing Cell Therapy in B-cell Acute Lymphocytic Leukemia(B-ALL)

The present Example describes, among other things, the identification ofnovel transcriptional gene signatures that predict patient relapse toCD19 CAR-expressing cell (e.g., T cell, NK cell) therapy (e.g., CTL019therapy) in B cell Acute Lymphocytic Leukemia (B-ALL), for use inaccordance with the present invention.

Among other things, the present Example describes novel gene signaturesbased on mRNA expression levels of selected genes in the patient priorto CD19 CAR-expressing cell (e.g., T cell, NK cell) treatment (e.g.,CTL019) (apheresis or bone marrow) or in manufactured CD19CAR-expressing cell (e.g., T cell, NK cell) product samples (e.g.,CTL019) prior to re-infusion. In an embodiment, the present exampledescribes novel gene signatures that discriminate relapsers to CTL019therapy in B-ALL from non-relapsers to CTL019 therapy in B-ALL.

The present Example describes methods of unbiased feature selection todiscover novel gene signatures that predict subject relapse to CD19CAR-expressing cell (e.g., T cell, NK cell) therapy (e.g., CTL019) inB-ALL, for use in accordance with the present invention.

The present Example also describes methods of Gene Set Analysis todiscover novel gene signatures, for use in accordance with the presentinvention.

Novel gene signatures based on mRNA expression levels in manufacturedCD19 CAR-expressing cell (e.g., T cell, NK cell) product samples priorto re-infusion were identified that predict subject relapse to CD19CAR-expressing cell (e.g., T cell, NK cell) therapy in B cell AcuteLymphocytic Leukemia (B-ALL). The identified signatures were discoveredin a whole genome RNAseq study of manufactured product samples whichincluded 7 B-ALL subject samples. B-ALL subject samples (7 total) werestratified as follows: biological samples were taken from 4 subjects whodid not relapse (“non-relapsers”) following CTL019 therapy, and 3subjects who did relapse (“relapsers”) following CTL019 therapy. Severalgene signatures discriminating responders from non-responders, andrelapsers from non-relapsers, in manufactured product samples werediscovered and are described further in detail below.

Novel gene signatures were then discovered using various data analyticalapproaches: 1) unbiased feature selection; 2) gene set analysis; and 3)differential expression analysis of selected genes of interest.

Novel gene signatures derived from unbiased feature selection werediscovered by determining which genes were differentially expressedbetween the 2-group comparison of relapsers and non-relapsers whichcompared the 3 relapsers to the 4 non-relapsers. Genes were defined asdifferentially expressed if their differential expression wasstatistically significant in the 2-group comparison with a FDR p-valuecutoff of 0.25. The gene list for the relapser versus non-relapsercomparison (N=17) is tabulated in Table 17. 2-group statistical modelswere applied to determine whether the meta-gene was statisticallydifferent between the groups, similar to the approach illustrated inFIG. 2B. FIG. 2B depicts an exemplary heat map of genes upregulated inactivated T_(EFF) versus resting T_(EFF) cells for complete responders(CR), partial responders (PR), and non-responders (NR).

Without wishing to be bound by a particular theory, these data indicatethat the differentiation state of T cells in CD19 CAR-expressing cell(e.g., T cell, NK cell) product (e.g., CTL019) correlate with subjectresponse (i.e., CR, PR, or NR) and predict subject relapse to CD19CAR-expressing cell (e.g., T cell, NK cell) therapy (e.g., CTL019therapy) in B-ALL. As described in Example 1, complete responders genesignatures are more like resting T_(REG) and T_(EFF) cells. Among otherthings, gene signatures for relapsers (e.g., a complete responder thatrelapses to CTL019 therapy) contain genes upregulated in T_(REG) versusT_(EFF) cells at resting. Without wishing to be bound by a particulartheory, these data indicate that relapsers to CAR-expressing cell (e.g.,T cell, NK cell) therapy (e.g., CTL019) in B-ALL have higher levels ofT_(REG) compared to non-relapsers to CAR-expressing cell (e.g., T cell,NK cell) therapy (e.g., CTL019). FIG. 21 depicts exemplary resultsillustrating that T_(REG) are differentially enriched in relapsers (R)versus non-relapsers, e.g., relapsers express high levels of T_(REG)genes compared to complete responders (CR) (e.g., non-relapsers). Anexemplary publication or sequence number disclosing the sequence of eachgene is also given in Table 17, and each publication is incorporated byreference in its entirety, including all nucleic acid and proteinsequences therein.

TABLE 17 Table 17: Exemplary Genes that Predict Patient Relapse toCTL019 Therapy Exemplary Gene miRBase Unigene Accession No. FDRPublication MIR199A1 MI0000242 NR_029586.1 2.11E−05 Landgraf et al.,Cell 129 (7), 1401-1414 (2007) PPIAL4D Hs.730589 NM_001164261.1 3.94E−05SEQ ID NO: 102 MIR1203 MI0006335 NR_031607.1 4.63E−03 Marton et al.,Leukemia 22 (2), 330- 338 (2008) uc021ovp 6.73E−03 SEQ ID NO: 103 ITM2CHs.111577 NM_001012514.2 1.17E−01 Yoshida et al., Int. J. NM_001012516.2Mol. Med. 25 (4), 649- NM_001287240.1 656 (2010) NM_001287241.1NM_030926.5 HLA-DQB1 Hs.409934 NM_001243961.1 1.17E−01 Pankuweit et al.,Gene Hs.534322 NM_001243962.1 531 (2), 180-183 NM_002123.4 (2013) TTTY10Hs.461175 NR_001542.1 1.25E−01 Derrien et al., Genome Res. 22 (9),1775-1789 (2012) TXLNG2P Hs.522863 NR_045128.1 2.27E−01 Prakash et al.,PLoS NR_045129.1 ONE 5 (10), E13284 (2010) MIR4650-1 MI0017277NR_039793.1 2.27E−01 Persson et al., Cancer Res. 71 (1), 78-86 (2011)KDM5D Hs.80358 NM_001146705.1 2.27E−01 Kim et al., J. Am. Soc.NM_001146706.1 Nephrol. 20 (9), 2025- NM_004653.4 2033 (2009) USP9YHs.598540 NM_004654.3 2.27E−01 Luddi et al., N. Engl. J. Med. 360 (9),881-885 (2009) PRKY Hs.584730 NR_028062.1 2.27E−01 Hogan et al., ClinMed Res 7 (3), 69-84 (2009) RPS4Y2 Hs.367761 NM_001039567.2 2.27E−01 Yeet al., BMC Bioinformatics 13, 134 (2012) RPS4Y1 Hs.282376 NM_001008.32.27E−01 Eljaafari et al., J. Immunol. 190 (1), 184-194 (2013)NCRNA00185 Hs.138453 NR_001543.3 2.28E−01 Prakash et al., PLoS Hs.729534NR_125733.1 ONE 5 (10), E13284 Hs.734681 NR_125734.1 (2010) NR_125735.1NR_125736.1 NR_125737.1 SULT1E1 Hs.479898 NM_005420.2 2.33E−01 Xu etal., Mol. Cell. Endocrinol. 369 (1-2), 140-149 (2013) EIF1AY Hs.461178NM_001278612.1 2.38E−01 Luna et al., NM_004681.3 Biochemistry 52 (52),9510-9518 (2013)

Gene set analysis yielded a number of gene signatures predictive ofsubject relapse to CTL019 therapy in B-ALL. The following genes showedincreased levels in relapsers and decreased levels in non relapsers:MIR199A1, MIR1203, uc021ovp, ITM2C, and HLA-DQB1. The following genesshowed decreased levels in relapsers and increased levels in nonrelapsers: PPIAL4D, TTTY10, TXLNG2P, MIR4650-1, KDM5D, USP9Y, PRKY,RPS4Y2, RPS4Y1, NCRNA00185, SULT1E1, and EIF1AY.

In particular, the present Example describes methods of Gene SetAnalysis to discover novel gene signatures, for use in accordance withthe present invention.

Among other things, the present Example describes novel gene signaturesbased on Gene Set Analysis, that are predictive of patent relapse toCD19 CAR-expressing cell (e.g., T cell, NK cell) therapy (e.g., CTL019)in B-ALL. Gene set analysis was performed on gene sets described inTable 17, and with gene sets described in Example 2, e.g., gene setswere sourced from (1) additional experiments were based on gene sets bySzabo et al., (disclosed herein); (2) gene sets published by Abbas etal. in Genome Research 2005; and (3) gene sets published by Gattinoni etal. in Nature Medicine 2011. Each of Szabo, Abbas and Gattinoni genesets are described in detail in Example 2. The gene sets defined bySzabo and considered in this analysis are tabulated in Table 2 ofExample 2. The gene sets defined by Abbas and considered in thisanalysis are tabulated in Table 3 of Example 2. The gene sets defined byGattinoni and considered in this analysis are tabulated in Table 4 ofExample 2.

Each gene set (e.g., B-ALL RNAseq gene sets, Szabo gene sets, Abbas genesets, and Gattinoni gene sets) was evaluated to determine itsassociation with subject response (i.e., relapser or non-relapser) inthe following manner: a meta-gene was calculated for each subject, wherethe meta-gene score for subject j was defined as

m _(j)=Σ_(i=G) ¹ x _(ij)−μ(x _(.j))/σ(x _(.j))

where x_(ij) is the expression value of gene i in subject j for a givengene set n=1, . . . , G; μ(x_(.j)) is the mean of genes 1, . . . , G insubject j; and σ(x_(.j)) is the standard deviation of genes 1, . . . , Gin subject j.

A 2-group statistical model was applied to each gene set to determinewhether the meta-gene was statistically different between themanufactured CTL019 product of relapsers and non-relapsers. A schematicillustrating this approach is given in FIG. 2B. Of the Szabo, Abbas, andGattinoni gene sets, there was one gene set that was significantlydifferentially enriched between relapsers and non-relapsers. This geneset was from the Szabo collection and contains genes upregulated inT_(REG) versus T_(EFF) cells at resting, and correlated with patientrelapse to CTL019 therapy. Specifically, this gene set was found to beenriched in relapsers, indicating that relapsers have higher levels ofT_(REGS) compared to non-relapsers. For example, the meta-gene score forthe gene set comprised of genes upregulated in T_(REG) in comparison toT_(EFF) cells is found to be correlated with patient relapse in productsamples (see FIG. 20). FIG. 20 depicts exemplary results (p=0.000215)illustrating that T_(REG) genes have high expression levels in relapsers(R) compared to non-relapser, complete responders (CR). The x-axis issamples by response group where CR=complete responder and R=relapser.The y-axis is normalized meta-gene expression scores.

In an embodiment, gene signatures described herein are used to enablemanufactured product improvements, thereby reducing the likelihood ofpatient relapse. In an embodiment, gene signatures described herein areused to modify therapeutic application of manufactured product, therebyreducing the likelihood of patient relapse.

In an embodiment, gene signatures described herein are identified in asubject prior to CAR-expressing cell (e.g., T cell, NK cell) treatment(e.g., a CD19 CAR-expressing cell treatment, e.g., CTL019 therapy) thatpredict relapse to CAR-expressing cell (e.g., T cell, NK cell)treatment. In an embodiment, gene signatures described herein areidentified in an apheresis sample. In an embodiment, gene signaturesdescribed herein are identified in a bone marrow sample. In anembodiment, gene signatures described herein are identified in amanufactured CAR-expressing cell (e.g., T cell, NK cell) product (e.g.,a CD19 CAR-expressing cell product, e.g., CTL019) prior to infusion.

Without wishing to be bound by a particular theory, these data indicatethat decreasing the T_(REG) signature in the patient prior to apheresisor during manufacturing of the CAR-expressing cell (e.g., T cell, NKcell) product significantly reduces the risk of patient relapse.

Example 7: Quantity of CD27+PD1− CART Cells Infused into PatientPredicts Response to Therapy

The number of CD27+PD1− cells in the CTL019 infusion product wasdetermined for 29 CLL patients (8 complete responders and 21non-responders). The relationship between number of CD27+PD1− CART cellsinfused and response to therapy is shown as a bar graph in FIG. 22. andas a scatter plot in FIG. 23. A threshold was set at 1×10⁷ CART cellsper patient. A statistically significant difference (p<0.0001) wasobserved between the complete responders and non-responders. Thisexperiment shows that complete remission of CLL patients to CART19immunotherapy is associated with higher numbers of infused CD27+PD1−CART cells.

EQUIVALENTS

Other embodiments of the invention will be apparent to those skilled inthe art from a consideration of the specification or practice of theinvention disclosed herein. While this invention has been disclosed withreference to specific aspects, it is apparent that other aspects andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all such aspectsand equivalent variations.

1. A method of evaluating a subject, having a cancer, comprising:acquiring a value of responder or relapser status to a therapycomprising a CAR-expressing cell population for the subject, whereinsaid value of responder or relapser status comprises a measure of one,two, three, four, five, six, seven, or more of the following: (i) thelevel or activity of CD27 and/or CD45RO− immune effector cells in asample; (ii) the level or activity of one, two, three, or more ofresting T_(EFF) cells, resting T_(REG) cells, younger T cells, or earlymemory T cells, or a combination thereof, in a sample; (iii) the levelor activity of one, two, three, or more of activated T_(EFF) cells,activated T_(REG) cells, older T cells, or late memory T cells, or acombination thereof, in a sample; (iv) the level or activity of animmune cell exhaustion marker in a sample; (v) the level or activity ofone, two, three, four, five, ten, twenty or more of the biomarkerslisted in Table 1A, Table 1B, Table 7A, Table 7B, Table 8, Table 9,Table 10, Table 14, Table 16, Table 17, Table 18, Table 20, FIG. 2B,PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L, KLRG1, or a CD19CAR-expressing cell gene set signature; (vi) a cytokine level oractivity in a CAR-expressing cell product sample, wherein the cytokineis chosen from one, two, three, four, five or more of the cytokineslisted in Table 16; (vii) a transduction efficiency of a CAR-expressingcell in a manufactured CAR-expressing cell product sample; or (viii) aquantity of CD27+PD-1− cells in a sample, wherein said value isindicative of the subject's responsiveness or relapsing status to theCAR-expressing cell therapy, thereby evaluating the subject. 2-3.(canceled)
 4. A method for treating a subject having a cancer,comprising: administering to the subject a therapeutically effectivedose of a CAR-expressing cell therapy, if the subject is identified asbeing responsive to a therapy comprising a CAR-expressing cellpopulation, wherein said identifying comprises a measure of one, two,three, four, five, six, seven, or more of the following: (i) the levelor activity of CD27 and/or CD45RO− immune effector cells in a sample;(ii) the level or activity of one, two, three, or more of restingT_(EFF) cells, resting T_(REG) cells, younger T cells, or early memory Tcells, or a combination thereof, in a sample; (iii) the level oractivity of one, two, three, or more of activated T_(EFF) cells,activated T_(REG) cells, older T cells, or late memory T cells, or acombination thereof, in a sample; (iv) the level or activity of animmune cell exhaustion marker in a sample; (v) the level or activity ofone, two, three, four, five, ten, twenty or more of the biomarkerslisted in Table 1A, Table 1B, Table 7A, Table 7B, Table 8, Table 9,Table 10, Table 14, Table 16, Table 17, Table 18, Table 20, FIG. 2B,PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L, KLRG1, or a CD19CAR-expressing cell gene set signature; (vi) a cytokine level oractivity in a CAR-expressing cell product sample, wherein the cytokineis chosen from one, two, three, four, five or more of the cytokineslisted in Table 16; or (vii) a transduction efficiency of aCAR-expressing cell in a manufactured CAR-expressing cell productsample; or (viii) a quantity of CD27+PD-1− cells in a sample, therebytreating the subject.
 5. The method of claim 4 wherein responsive tosaid value, performing one, two, three, four, five, six, seven, or moreof: identifying the subject as a complete responder, partial responderor non-responder, or a relapser or a non-relapser; administering aCAR-expressing cell therapy; administering an altered dosing of aCAR-expressing cell therapy; altering the schedule or time course of aCAR-expressing cell therapy; administering an additional agent incombination with a CAR-expressing cell therapy; administering to anon-responder or partial responder a therapy that increases the numberof younger T cells or naïve T cells in the subject prior to treatmentwith a CAR-expressing cell therapy; modifying a manufacturing process ofa CAR-expressing cell therapy; administering an alternative therapy; orif the subject is, or is identified as, a non-responder or a relapser,decreasing the T_(REG) cell population and/or T_(REG) gene signature. 6.The method of claim 4, wherein the CAR-expressing cell therapy comprisesa plurality of CAR-expressing immune effector cells.
 7. The method ofclaim 4, wherein the CAR-expressing cell therapy is a CAR19 therapy. 8.The method of claim 4, wherein the measure of one or more of (i)-(viii)is obtained from: (a) an apheresis sample acquired from the subject,wherein optionally the apheresis sample; or (b) a manufacturedCAR-expressing cell product sample.
 9. (canceled)
 10. The method ofclaim 4, wherein the subject is evaluated prior to, during, or afterreceiving the CAR-expressing cell therapy.
 11. The method of claim 4,wherein the cancer is a hematological cancer.
 12. The method of claim 4,wherein the hematological cancer is an ALL or a CLL.
 13. The method ofclaim 4, wherein the subject is a human patient.
 14. The method of claim4, further comprises identifying the subject as a responder, anon-responder, a relapser or a non-relapser, based on a measure of oneor more of (i)-(viii).
 15. The method of claim 4, wherein the measure ofone or more of (i)-(viii) evaluates a profile for one or more of geneexpression, flow cytometry or protein expression.
 16. The method ofclaim 4, wherein the level or activity of a CD8+ T cell is evaluatedusing a profile or signature indicative of the percentage of CD8+ T cellin the sample.
 17. The method of claim 4, wherein the level or activityof CD27+CD45RO− immune effector cells is evaluated using a profile orsignature indicative of the percentage of CD27+CD45RO− immune effectorcells in the sample.
 18. The method of claim 4, wherein the level oractivity is evaluated using a profile or gene signature according toone, two, three, four, five, ten, twenty, fifty, sixty, seventy, onehundred or more of a biomarker or gene set listed in Tables 1A, 1B, 3,4, 5, 6, 7A, 7B, or FIG. 2B.
 19. The method of claim 4, wherein thebiomarker is a secreted or a cell surface biomarker listed in Table 8.20. The method of claim 4, wherein a responder has, or is identified ashaving, a greater level or activity of one, two, or more of GZMK,PPF1BP2, or naïve T cells as compared to a non-responder.
 21. The methodof claim 4, wherein a non-responder has, or is identified as having, agreater level or activity of one, two, three, four, five, six, seven, ormore of IL22, IL-2RA, IL-21, IRF8, IL8, CCL17, CCL22, effector T cells,or regulatory T cells, as compared to a responder.
 22. The method ofclaim 4, wherein a complete responder has, or is identified as having:(a) a greater percentage of CD8+ T cells compared to a reference value;(b) a greater percentage of CD27+CD45RO− immune effector cells comparedto a reference value; (c) a greater percentage of CD4+ T cells comparedto a reference value; or (d) a greater percentage of one, two, three, ormore of resting TEFF cells, resting TREG cells, younger T cells, orearly memory T cells, or a combination thereof, compared to a referencevalue. 23-25. (canceled)
 26. The method of claim 4, wherein anon-responder has, or is identified as having: (a) a greater percentageof one, two, three, or more of activated TEFF cells, activated TREGcells, older T cells, or late memory T cells, or a combination thereof,compared to a reference value, e.g., a responder number of activatedTEFF cells, activated TREG cells, older T cells, or late memory T cells;or (b) a greater percentage of PD-1 or LAG-3 expressing immune effectorcells compared to the percentage of PD-1 or LAG-3 expressing immuneeffector cells from a responder, or a greater percentage of PD-1+/LAG-3+cells in the CAR-expressing cell population compared to a responder tothe CAR-expressing cell therapy, an exhausted phenotype of PD1+CAR+ andco-expression of LAGS in the CAR-expressing cell population, or agreater percentage of PD-1+/TIM-3+ cells in the CAR-expressing cellcompared to the responder.
 27. (canceled)
 28. The method of claim 4,wherein a partial responder has, or is identified as having, a higherpercentage of PD-1+/LAG-3+ cells than responders in the CAR-expressingcell population, or a higher percentage of PD-1+/TIM-3+ cells thanresponders in the CAR-expressing cell population.
 29. The method ofclaim 4, wherein the presence of CD8+CD27+CD45RO− T cells in anapheresis sample is a positive predictor of the subject response to aCAR-expressing cell therapy.
 30. The of claim 4, wherein a highpercentage of PD1+CAR+ and LAG3+ or TIM3+ T cells in an apheresis sampleis a poor prognostic predictor of the subject response to aCAR-expressing cell therapy.
 31. The method of claim 4, wherein aresponder to a CAR19 therapy has, or is identified as having, thebiomarker profile of Table
 9. 32. The method of claim 4, wherein anon-responder to a CAR19 therapy has, or is identified as having: (a)the biomarker comprising one or more of PD-1+ immune effector cells,TIM-3+ immune effector cells, LAG-3+ immune effector cells, KLRG1+immune effector cells, CD27-immune effector cells, activated TEFF cells,activated TREG cells, activated TH1, activated TH2 cells, stimulatedmemory cells, or late T memory cells, or a combination thereof; or (b)the biomarker profile of Table
 10. 33. (canceled)
 34. The method ofclaim 4, wherein gene expression of one, two, three, four or more ofKLRG1, CD57, CD27, CD122, or CD62L is predictive of patient response toCTL019 therapy.
 35. The method of claim 4, wherein a relapser has, or isidentified as having, elevated levels of one, two, three, four, five,six, seven, eight, nine, ten or more of C5orf32, CCL17, CSF1, CTSL1,EMP1, EPAS1, GCLM, GK, GPR56, HMOX1, IKBIP, IL10, IL13, IL1RN, IL4, IL5,IL9, MIR155, PANX2, PGAM4, PRKAR1B, TNFRSF11A, TNFRSF1B, TNFRSF8,VTRNA1-3, or ZNF282.
 36. The method of claim 4, wherein the responderhas one, two, three or more of the following profile: (i) has a greaternumber of CD27+ immune effector cells compared to a reference value;(ii) has a greater number of CD8+ T cells compared to a reference value;(iii) has a lower number of cells expressing one or more checkpointinhibitors compared to a reference value; or (iv) has a greater numberof one, two, three, or more of resting TEFF cells, resting TREG cells,younger cells, or early memory T cells, or a combination thereof,compared to a reference value.
 37. The method of claim 4, wherein thecytokine level or activity of (vi) is chosen from one, two, three, four,five, six, seven, eight, or more of cytokine CCL20/MIP3a, IL17A, IL6,GM-CSF, IFNγ, IL10, IL13, IL2, IL21, IL4, IL5, IL9 or TNFα, or acombination thereof.
 38. The method of claim 4, wherein a transductionefficiency of 15% or higher in (vii) is indicative of increasedresponsiveness or decreased relapse.
 39. The method of claim 4, whereinsaid value of responder or relapser status comprises a measure of thelevel or activity of one, two, three, four, five, ten, twenty or more ofthe biomarkers having a FDR p-value of below 0.1 or 0.01 listed in Table1A, 1B, 17, 18, or
 20. 40. A method of evaluating the potency of aCAR-expressing cell product, said method comprising: acquiring a valuefor one, two, three, four, five, six, seven, eight, or more of: (i) thelevel or activity of CD27 and/or CD45RO− immune effector cells in theCAR-expressing cell product; (ii) the level or activity of one, two,three, or more of resting T_(EFF) cells, resting T_(REG) cells, youngerT cells, or early memory T cells, or a combination thereof, in theCAR-expressing cell product; (iii) the level or activity of one, two,three, or more of activated T_(EFF) cells, activated T_(REG) cells,older T cells, or late memory T cells, or a combination thereof, in theCAR-expressing cell product; (iv) the level or activity of an immunecell exhaustion marker; (v) the level or activity of one, two, three,four, five, ten, twenty or more of the biomarkers listed in Table 1A,Table 1B, Table 7A, Table 7B, Table 8, Table 9, Table 10, Table 14,Table 16, Table 17, Table 18, Table 20, FIG. 2B, PD-1, LAG-3, TIM-3,CD57, CD27, CD122, CD62L, KLRG1, or a CD19 CAR-expressing cell gene setsignature; (vi) a cytokine level or activity in the CAR-expressing cellproduct sample wherein the cytokine is chosen from one, two, three,four, five or more of the cytokines listed in Table 16; (vii) atransduction efficiency of CAR-expressing cells in the product; (viii) aquantity of CD27+PD-1− cells in a sample; or (ix) the level or activityof a T_(REG) cell or cell population; wherein: (a) an increase in (i),(ii), (vi), (vii), (viii), or any combination thereof, is indicative ofincreased potency of the CAR-expressing cell product, and wherein anincrease in (iii), (iv), (ix), or any combination thereof, is indicativeof decreased potency of the CAR-expressing cell product; (b) thecytokine is chosen from one, two, three, four, five, six, seven, eight,or more (or all) of CCL20/MIP3a, IL17A, IL6, GM-CSF, IFNγ, IL10, IL13,IL2, IL21, IL4, IL5, IL9 or TNFα, or a combination thereof; or (c) atransduction efficiency of 15% or higher is indicative of increasedpotency. 41-42. (canceled)
 43. A method for optimizing manufacturing ofa CAR-expressing cell product comprising: (1) acquiring a samplecomprising CAR-expressing cell; (2) activating the CAR-expressing cellin vitro; (3) evaluating the potency of the potency of the activatedCAR-expressing cell by determining one, two, three, four, five, six,seven, eight, or more of: (i) the level or activity of CD27 and/orCD45RO− immune effector cells in the CAR-expressing cell product; (ii)the level or activity of one, two, three, or more of resting T_(EFF)cells, resting T_(REG) cells, younger T cells, or early memory T cells,or a combination thereof, in the CAR-expressing cell product; (iii) thelevel or activity of one, two, three, or more of activated T_(EFF)cells, activated T_(REG) cells, older T cells, or late memory T cells,or a combination thereof, in the CAR-expressing cell product; (iv) thelevel or activity of an immune cell exhaustion marker in theCAR-expressing cell product; (v) the level or activity of one, two,three, four, five, ten, twenty or more of the biomarkers listed in Table1A, Table 1B, Table 7A, Table 7B, Table 8, Table 9, Table 10, Table 14,Table 16, Table 17, Table 18, Table 20, FIG. 2B, PD-1, LAG-3, TIM-3,CD57, CD27, CD122, CD62L, KLRG1, or a CD19 CAR-expressing cell gene setsignature; (vi) a cytokine level or activity in a CAR-expressing cellproduct sample, wherein the cytokine is chosen from one, two, three,four, five or more of the cytokines listed in Table 16; (vii) atransduction efficiency of CAR-expressing cells in the product; (viii) aquantity of CD27+PD-1− cells in a sample; or (ix) the level or activityof a T_(REG) cell or cell population, wherein an increase in (i), (ii),(vi), (vii), (viii), or any combination thereof, is indicative ofincreased potency of the CAR-expressing cell product, and wherein anincrease in (iii), (iv), (ix), or any combination thereof, is indicativeof decreased potency of the CAR-expressing cell product. therebyoptimizing manufacturing of the product.
 44. The method of claim 43,further comprising a step of enriching for cells having an increase inany of (i), (ii), (vi), (vii), (viii), or any combination thereof, or adecrease in any of (iii), (iv), (ix), or any combination thereof; orwherein the cytokine level or activity is chosen from one or more ofcytokine CCL20/MIP3a, IL17A, IL6, GM-CSF, IFNγ, IL10, IL13, IL2, IL21,IL4, IL5, IL9 or TNFα, or a combination thereof.
 45. (canceled)
 46. Themethod of claim 43, which comprises a further step that depletes T_(REG)cells.
 47. The method of claim 46, wherein T_(REG) cells are depletedvia CD25-depletion, GITR depletion, mTOR inhibition, or a combinationthereof.
 48. The method of claim 43, wherein (i), (ii), (iii), (iv),(v), (vi), (vii), (viii), (ix), or any combination thereof (e.g., all)are evaluated following activation in vitro, and/or wherein theCAR-expressing cell therapy comprises CTL019.
 49. (canceled)
 50. Themethod of claim 4, wherein the subject receives a treatment prior to theinitiation of a CAR-expressing cell therapy or a treatmentpost-CAR-expressing cell therapy.
 51. The method of claim 4, wherein thecancer is associated with CD19 expression.
 52. The method of claim 4,wherein the cancer is a hematological cancer.
 53. The method of claim 4,wherein the cancer or hematological cancer is selected from the groupconsisting of B-cell acute lymphocytic leukemia (B-ALL), T-cell acutelymphocytic leukemia (T-ALL), acute lymphocytic leukemia (ALL), chronicmyelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), B cellpromyelocytic leukemia, blastic plasmacytoid dendritic cell neoplasm,Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma,hairy cell leukemia, small cell- or a large cell-follicular lymphoma,malignant lymphoproliferative conditions, MALT lymphoma, mantle celllymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia andmyelodysplastic syndrome, non-Hodgkin lymphoma, Hodgkin lymphoma,plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, andWaldenstrom macroglobulinemia.
 54. The method of claim 4, where the CD19CAR-expressing cell gene signature comprises a value for expression ofat least 5, 6, 7, 8, 9 or 10 genes comprising a CD19 CAR-expressing cellgene signature.
 55. A kit for providing a prognosis for success rate ofa CAR-expressing cell therapy in a subject having cancer, said kitcomprising: a set of reagents that specifically detects the level oractivity of one or more genes listed in Table 1A, Table 1B, Table 7A,Table 7B, Table 8, Table 9, Table 10, Table 14, Table 17, Table 18,Table 20, FIG. 2B, PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L, KLRG1,and/or a CD19 CAR-expressing cell gene set signature; and instructionsfor using said kit; wherein said instructions for use provide that ifone or more of the detected expression levels is different from thesubject is more likely to respond positively to a CAR-expressing celltherapy; and wherein the set of reagent detects the expression of mRNAexpressed from said set of genes; and/or wherein the set of reagentsdetects the expression of polypeptides encoded by said set of genes.56-57. (canceled)
 58. A system for evaluating cancer in a subject,comprising: at least one processor operatively connected to a memory,the at least one processor when executing is configured to: acquire avalue of responder or relapser status that comprises a measure of one,two, three, four, five, six, seven, or more of the following: (i) thelevel or activity of CD27 and/or CD45RO− immune effector cells; (ii) thelevel or activity of one, two, three, or more of resting T_(EFF) cells,resting T_(REG) cells, younger T cells, or early memory T cells, or acombination thereof, in a sample; (iii) the level or activity of one,two, three, or more of activated T_(EFF) cells, activated T_(REG) cells,older T cells, or late memory T cells, or a combination thereof, in asample; (iv) the level or activity of an immune cell exhaustion marker;(v) the level or activity of one, two, three, four, five, ten, twenty ormore of the biomarkers listed in Table 1A, Table 1B, Table 7A, Table 7B,Table 8, Table 9, Table 10, Table 14, Table 16, Table 17, Table 18,Table 20, FIG. 2B, PD-1, LAG-3, TIM-3, CD57, CD27, CD122, CD62L, KLRG1,or a CD19 CAR-expressing cell gene set signature; (vi) a cytokine levelor activity in a CAR-expressing cell product sample, wherein thecytokine is chosen from one, two, three, four, five or more of thecytokines listed in Table 16; (vii) a transduction efficiency of aCAR-expressing cell in a manufactured CAR-expressing cell productsample; or (viii) a quantity of CD27+PD-1− cells in a sample, responsiveto a determination of the value of responder status, perform one, two,three, four, five, six, seven, or more of: identify the subject as acomplete responder, partial responder, non-responder, relapser ornon-relapser; recommend administering a CAR-expressing cell therapy;recommend a selection or alteration of a dosing of a CAR-expressing celltherapy; recommend a selection or alteration of a schedule or timecourse of a CAR-expressing cell therapy; recommend administering anadditional agent in combination with a CAR-expressing cell therapy;recommend administering to a non-responder or partial responder atherapy that increases the number of naïve T cells in the subject priorto treatment with a CAR-expressing cell therapy; recommend modifying amanufacturing process of a CAR-expressing cell therapy; recommendmodifying the CAR-expressing cell product prior to infusion into thepatient; recommend adjusting the CAR-expressing cell infusion dose toachieve clinical efficacy; recommend administering an alternativetherapy; recommend a selection of an alternative therapy; or if thesubject is, or is identified as, a non-responder or a relapser,recommend decreasing the T_(REG) cell population and/or T_(REG) genesignature administration of cyclophosphamide, an anti-GITR antibody, anmTOR inhibitor, or a combination thereof. 59-64. (canceled)
 65. Themethod of claim 4, wherein the CAR19 therapy is CTL019.
 66. The methodof claim 4, wherein the immune effector cells are in a CD4+ or a CD8+ Tcell population.
 67. The method of claim 4, wherein the younger T cellsare younger CD4 or CD8 cells, or gamma/delta cells.
 68. The method ofclaim 4, wherein the immune cell exhaustion marker is one, two or moreimmune checkpoint inhibitors.
 69. The method of claim 68, wherein theimmune checkpoint inhibitor is PD-1, TIM-3 and/or LAG-3.
 70. The methodof claim 4, wherein the biomarker is CCL20, IL-17a and/or IL-6.
 71. Themethod of claim 4, wherein the cytokine level or activity is a qualityof cytokine repertoire.
 72. The method of claim 4, wherein the quantityof CD27+PD-1− cells in a sample is a quantity greater than or equal to1×10⁷ cells.
 73. The method of claim 8, wherein the apheresis sample orthe manufactures CAR-expressing cell product is evaluated prior toinfusion or re-infusion.
 74. The method of claim 22, wherein thereference value is a value of a non-responder percentage of CD8+ Tcells.